Cell-targeting molecules comprising de-immunized, shiga toxin a subunit effectors and cd8+ t-cell epitopes

ABSTRACT

The present invention provides cell-targeting molecules which can deliver a CD8+ T-cell epitope cargo to the MHC class I presentation pathway of a target cell. The cell-targeting molecules of the invention can be used to deliver virtually any CD8+ T-cell epitope from an extracellular space to the MHC class I pathway of a target cell, which may be a malignant cell and/or non-immune cell. The target cell can then display on a cell-surface the delivered CD8+ T-cell epitope complexed with MHC I molecule. The cell-targeting molecules of the invention have uses which include the targeted labeling and/or killing of specific cell-types within a mixture of cell-types, including within a chordate, as well as the stimulation of beneficial immune responses. The cell-targeting molecules of the invention have uses, e.g., in the treatment of a variety of diseases, disorders, and conditions, including cancers, tumors, growth abnormalities, immune disorders, and microbial infections.

TECHNICAL FIELD

The present invention relates to cell-targeting molecules which eachcomprise (1) a binding region for cell-targeting, (2) a Shiga toxin ASubunit effector polypeptide for subcellular delivery, and (3) one ormore, CD8+ T-cell epitopes which is heterologous to the Shiga toxin ASubunit effector polypeptide; wherein the cell-targeting molecule iscapable of delivering at least one, heterologous, CD8+ T-cell epitope tothe MHC class I presentation pathway of a target cell, such as, e.g. amalignant cell. The Shiga toxin effector polypeptide components of thecell-targeting molecules of the present invention comprise a combinationof mutations relative to a wild-type Shiga toxin sequence providing (1)de-immunization. (2) a reduction in protease sensitivity, and/or (3) anembedded, T-cell epitope(s); wherein each Shiga toxin effectorpolypeptide retains one or more Shiga toxin function, such as, e.g.,stimulating cellular internalization, directing intracellular routing,and/or potent cytotoxicity. In certain embodiments, the cell-targetingmolecule of the present invention can deliver the heterologous, CD8+T-cell epitope to the MHC class I presentation pathway of a target cellwherein the heterologous, CD8+ T-cell epitope is linked, either directlyor indirectly, carboxy-terminal to the carboxy-terminus of a Shiga toxinA1 fragment derived region of the Shiga toxin A Subunit effectorpolypeptide. In certain embodiments, the cell-targeting molecules of thepresent invention are useful for administration to chordates, such as,e.g., when it is desirable to (1) reduce or eliminate a certain immuneresponse(s) resulting from the administered molecule, (2) reduce oreliminate non-specific toxicities resulting from the administeredmolecule, (3) specifically kill a target cell(s) in vivo, and/or (4)target a beneficial immune response(s) to a target cell-type, a tumormass comprising a target cell-type, and/or a tissue locus comprising atarget cell-type, such as via stimulating intercellular engagement of aCD8+ T-cell(s) of the chordate with the target cell-type. Thecell-targeting molecules of the present invention have numerous uses,e.g., for the delivery of a specific CD8+ T-cell epitope from anextracellular location to the MHC class I presentation pathway of atarget cell; the cell-surface labeling of a target cell with a MHC classI displayed CD8+ T-cell epitope; the selective killing of specificcell-types in the presence of other cells; the stimulation of beneficialimmune responses in vivo; the elicitation of a cytotoxic T lymphocytecell response(s) to a target cell; the repression of a detrimentalimmune response(s) in vivo; the creation of memory immune cells, and thediagnosis and treatment of a variety of diseases, disorders, andconditions, such as, e.g., cancers, tumors, other growth abnormalities,immune disorders, and microbial infections.

BACKGROUND

The following includes information that may be useful in understandingthe invention(s) described herein. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently described or claimed invention(s), or that any publication ordocument that is specifically or implicitly referenced herein is priorart.

The concept of harnessing the power of the immune system to treat canceris over one hundred years old (see e.g. Wiemann B, Starnes C, PharmacolTher 64: 529-64 (1994)). For example, William Cole) used patients'reactions to inactivated infectious agents to improve their immunedefenses to cancers. By triggering infection-like immune reactions, thepatient's immune system became stimulated and showed improvedimmunosurveillance of cancer cells, often leading to disease remission.It may be possible to improve upon this concept by using targetedtherapeutics and by limiting or focusing immune reactions to localizedareas, such as, e.g., via the exquisite specificity of the adaptiveimmune system. Targeted therapies may be used to target cancer cellsand/or cancer tissue loci within a patient for the receipt of highlyimmunogenic, foreign epitopes (e.g., from an infectious agent) in orderto locally activate a variety of beneficial immune responses and tospecifically mark cancer cells as being foreign using epitopes which aremore immunogenic than any already displayed by the cancer cells in thepatient. Further, by inducing an imitation of an infected state for alarge number of targeted cancer cells, the patient's immune system maybecome systemically stimulated such as, e.g., including an increase inglobal immunosurveillance and immune cell activity, but the exquisitespecificity of the immune system may limit or focus immune responses toa certain tissue location, cell-type(s), or even a single foreignepitope(s). This approach may activate the immune system generally in asystemic way (such as shown with Coley's toxins, cytokine therapies,immune-checkpoint inhibitors, and cancer vaccines) while focusingbeneficial immune responses to certain tissues and cells in a localizedway (such as shown with adoptive chimeric antigen receptor-engineered Tcell (CAR-T) and tumor-targeted monoclonal antibody therapies).

The major histocompatibility (MHC) class I system plays an essentialrole in the immune system by providing epitope presentation ofintracellular antigens (Cellular and Molecular Immunology (Abbas A, ed.,Saunders, 8^(th) ed., 2014)). This process is thought to be an importantpart of the adaptive immune system, a system which evolved in chordatesprimarily to protect against intracellular pathogens as well asmalignant cells expressing intracellular antigens, such as, e.g., cancercells. For example, human infections involving intracellular pathogensmay only be overcome by the combined actions of both the MHC class I andclass II systems (see e.g. Chiu C, Openshaw P, Nat Immunol 16: 18-26(2015)). The MHC class 1 system's contribution is to identify and killmalignant cells based on the identification of intracellular antigens.

The MHC class I system functions in any nucleated cell of a vertebrateto present intracellular (or endogenous) antigens, whereas the MHC classII pathway functions in professional antigen-presenting cells (APCs) topresent extracellular (or exogenous) antigens (Neefjes J et al., Nat RevImmunol 11; 823-36 (2011)). Intracellular or “endogenous” epitopesrecognized by the MHC class I system are typically fragments ofmolecules encountered in the cytosol or lumen of the endoplasmicreticulum (ER) of a cell, and these molecules are typicallyproteolytically processed by the proteasome and/or another protease(s)in the cytosol. When present in the ER, these endogenous epitopes areloaded onto MHC class I molecules and presented on the surface of thecell as peptide-MHC class I molecule complexes (pMHC Is). In contrast,the MHC class II system functions only in specialized cells to recognizeexogenous epitopes derived from extracellularly encountered moleculesprocessed only in specific endosomal compartments, such as, e.g., lateendosomes, lysosomes, phagosomes, and phagolysosomes, and includingintracellular pathogens residing in endocytotic organelles.

The presentation of specific epitope-peptides complexed with MHC class Imolecules by nucleated cells in chordates plays a major role instimulating and maintaining immune responses to intracellular pathogens,tumors, and cancers. Intercellular CD8+ T lymphocyte (T-cell) engagementof a cell presenting a specific epitope-MHC class I complex by a CD8+T-cell initiates protective immune responses that can result in therejection of the presenting cell, i.e. death of the presenting cell dueto the cytotoxic activity of one or more cytotoxic T lymphocytes (CTLs).The specificity of this intercellular engagement is determined bymultiple factors, CD8+ T-cells recognize pMHC Is on the cell surface ofanother cell via their TCRs, CD8+ T-cells express different T-cellreceptors (TCRs) with differing binding specificities to differentcognate pMHC Is, CD8+ T-cell specificity depends on each individualT-cell's specific TCR and that TCR's binding affinity to the presentedepitope-MHC complex as well as the overall TCR binding occupancy to thepresenting cell. In addition, there are diverse variants of MHC class Imolecules that influence intercellular CD8+ T-cell recognition in atleast in two ways; by affecting the specificity of peptides loaded anddisplayed (i.e. the pMHC I repertoire) and by affecting the contactregions between TCRs and pMHC Is involved in epitope recognition.

The presentation of certain epitopes complexed with MHC class Imolecules can sensitize the presenting cell to targeted killing bylysis, induced apoptosis, and/or necrosis. CTL killing of pMHCI-presenting cells occurs primarily via cytolytic activities mediated bythe delivery of perform and/or granzyme into the presenting cell viacytotoxic granules (see e.g. Russell J, Ley T, Annu Rev Immunol 20;323-70 (2002); Cullen S. Martin S, Cell Death Dif 15; 251-62 (2008)).Other CTL-mediated target cell killing mechanisms involve inducingapoptosis in the presenting cell via TNF signaling, such as, e.g., viaFasL/Fas and TRAIL/TRAIL-DR signaling (see e.g. Topham D et al., JImmunol 159; 5197-200 (1997); Ishikawa E et al., J Virol 79; 7658-63(2005); Brincks E et al., J Immunol 181; 4918-25 (2008); Cullen S,Martin S, Cell Death Diff 15; 251-62 (2008)). Furthermore, activatedCTLs can indiscriminately kill other cells in proximity to therecognized, pMHC I-presenting cell regardless of the peptide-MHC class Icomplex repertoires being presented by the other proximal cells(Wiedemann A et al., Proc Natl Acad Sci USA 103; 10985-90 (2006)). Inaddition, activated CTLs can release immuno-stimulatory cytokines,interleukins, and other molecules to influence the immuno-activation ofthe microenvironment.

This MHC class I and CTL immunosurveillance system could conceivably beharnessed by certain therapies to guide a subject's adaptive immunesystem into rejecting and specifically killing certain cell types. Inparticular, the MHC class I presentation pathway may be exploited byvarious therapeutic molecules to force certain targeted cells to displaycertain epitopes on cell surfaces in order to induce desired immuneresponses including the increasing immuno-detection and killing ofspecifically targeted cells by immune cells. Therapeutic molecules mightbe designed which specifically deliver CD8+ T-cell epitopes to the MHCclass I pathway for presentation by malignant cells (e.g. tumor orinfected cells) to signal their own destruction and, perhaps, in theaggregate to educe a more wide-spread stimulation of the immune system.In addition, therapeutic molecules might be designed which alsostimulate or increase MHC presentation activity in target cells.

It would be desirable to have cell-targeting molecules capable, whenexogenously administered, of delivering a CD8+ T-cell epitope to the MHCclass I presentation pathway of a chosen target cell, where the epitopemay be chosen form a wide variety of epitopes, such as, e.g., fromcommon infectious agents, and the target cell may be chosen from a widevariety of cells, such as, e.g., malignant and/or infected cells,particularly cells other than professional APCs like dendritic cells.Such cell-targeting molecules, which preferentially target malignantcells over healthy cells, may be administered to a chordate for in vivodelivery of a CD8+ T-cell epitope for MHC class I presentation by targetcells, such as, e.g., infected, neoplastic, or otherwise malignantcells. In addition, it may be desirable in certain circumstances to havesuch cell-targeting molecules that also directly kill target cells via anon-immune system based mechanism. Furthermore, it would be desirable tohave such cell-targeting molecules that also exhibit low antigenicity,low immunogenicity, high stability, and/or low non-specific toxicityafter administration to a chordate.

SUMMARY OF THE INVENTION

The present invention provides various embodiments of cell-targetingmolecules, and compositions thereof, wherein each cell-targetingmolecule comprises 1) at least one Shiga toxin A Subunit effectorpolypeptide derived from the A Subunit of at least one member of theShiga toxin family, 2) at least one binding region capable ofspecifically binding at least one extracellular target biomolecule, and3) at least one CD8+ T-cell epitope-peptide cargo; and wherein eachcell-targeting molecule is capable of delivering, from an extracellularlocation, the at least one CD8+ T-cell epitope-peptide to the MHC classI presentation pathway of a cell. For each cell-targeting molecule ofthe present invention, the at least one binding region is heterologousto the Shiga toxin A Subunit effector polypeptide. For certainembodiments of the cell-targeting molecule of the present invention, theat least one Shiga toxin effector polypeptide (i) has a Shiga toxin A1fragment derived region having a carboxy-terminus, (ii) comprises adisruption of at least one, endogenous, B-cell and/or CD4+ T-cellepitope region, and (iii) comprises a disrupted furin-cleavage motif atthe carboxy-terminus of the A1 fragment derived region. For eachcell-targeting molecule of the present invention, the at least one CD8+T-cell epitope-peptide cargo is (i) heterologous to the Shiga toxin ASubunit effector polypeptide and (ii) not embedded or inserted in theShiga toxin A1 fragment region and/or the Shiga toxin A Subunit effectorpolypeptide (see e.g. FIG. 1, depicting illustrative examples ofexemplary embodiments of cell-targeting molecules of the presentinvention).

For certain embodiments of the cell-targeting molecule of the presentinvention, upon administration of the cell-targeting molecule to a cellresults in (i) the internalization of the cell-targeting molecule by thecell and (ii) the cell presenting on a cellular surface the CD8+ T-cellepitope-peptide cargo complexed with a MHC class I molecule.

For certain embodiments of the cell-targeting molecule of the presentinvention, upon administration of the cell-targeting molecule to a cell,which is physically coupled with extracellular target biomolecule boundby the binding region of the cell-targeting molecule, results in thecell presenting on a cellular surface the CD8+ T-cell epitope-peptidecargo complexed with a MHC class I molecule. In certain furtherembodiments, having or placing the cell in the presence of an immunecell(s) further results in an immune cell response in trans, aninter-cellular engagement of the cell by an immune cell (e.g. acytotoxic T lymphocyte), and/or death of the cell induced via aninter-cellular action(s) of an immune cell.

For certain embodiments of the cell-targeting molecule of the presentinvention, upon administration of the cell-targeting molecule to achordate, which comprises cells physically coupled with extracellulartarget biomolecule bound by the binding region of the cell-targetingmolecule, results in at least some of said cells presenting on acellular surface the CD8+ T-cell epitope-peptide cargo complexed with aMHC class I molecule. In certain further embodiments, the resultsfurther include an immune cell response in trans, such as, e.g., theinter-cellular engagement of at least some of said cells by an immunecell and/or death of the cell induced via an inter-cellular action(s) ofan immune cell (e.g. a cytotoxic T lymphocyte).

Cell-targeting molecules of the present invention may be used fortargeted delivery of various CD8+ T-cell epitopes to any nucleated,target cell within a chordate in order to cause the delivered CD8+T-cell epitope-peptide cargo to be presented on the target cell surfacecomplexed with a MHC class I molecule. The target cells can be ofvarious types, such as, e.g., neoplastic cells, infected cells, cellsharboring intracellular pathogens, and other undesirable cells, and thetarget cell can be targeted by cell-targeting molecules of the inventioneither in vitro or in vivo. In addition, the present invention providesvarious cell-targeted molecules comprising protease-cleavage resistant,Shiga toxin effector polypeptides capable of intracellular delivery ofheterologous, CD8+ T-cell epitopes to the MHC class I presentationpathways of target cells while simultaneously improving extracellular,in vivo tolerability of these cell-targeting molecules. Certaincell-targeting molecules of the present invention have improvedusefulness for administration to chordates as either a therapeuticand/or diagnostic agent because of the reduced likelihood of producingnonspecific toxicities at a given dosage.

In certain embodiments of the cell-targeting molecule of the presentinvention, the CD8+ T-cell epitope-peptide cargo is fused, eitherdirectly or indirectly, to the Shiga toxin A Subunit effectorpolypeptide and/or the binding region. In certain further embodiments ofthe cell-targeting molecule of the present invention, the CD8+ T-cellepitope-peptide cargo is fused via a peptide bond, either directly orindirectly, to the Shiga toxin A Subunit effector polypeptide and/or thebinding region. In certain further embodiments, the CD8+ T-cellepitope-peptide cargo is fused via a peptide bond, either directly orindirectly, to the Shiga toxin A Subunit effector polypeptide and/or thebinding region as a genetic fusion.

In certain embodiments, the cell-targeting molecule of the presentinvention comprises a polypeptide comprising the binding region, theShiga toxin effector polypeptide, and the CD8+ T-cell epitope-peptidecargo.

In certain embodiments, the cell-targeting molecule of the presentinvention comprises the binding region comprising two or morepolypeptide chains and the CD8+ T-cell epitope-peptide cargo is fused toa polypeptide comprising the Shiga toxin effector polypeptide and one ofthe two or more polypeptide chains.

In certain embodiments of the cell-targeting molecule of the presentinvention, the CD8+ T-cell epitope-peptide cargo is positionedcarboxy-terminal to the carboxy terminus of the Shiga toxin A1 fragmentderived region.

In certain embodiments, the cell-targeting molecule of the presentinvention comprises a molecular moiety associated with thecarboxy-terminus of the Shiga toxin A Subunit effector polypeptide. Incertain further embodiments, the molecular moiety comprises the bindingregion.

In certain embodiments, the cell-targeting molecule of the presentinvention comprises a molecular moiety associated with thecarboxy-terminus of the Shiga toxin A Subunit effector polypeptidewherein the molecular moiety is cytotoxic.

In certain embodiments, the cell-targeting molecule of the presentinvention comprises a molecular moiety which comprises at least oneamino acid and the Shiga toxin A Subunit effector polypeptide is linkedto at least one amino acid residue of the molecular moiety. In certainfurther embodiments, the molecular moiety and the Shiga toxin A Subuniteffector polypeptide are fused forming a continuous polypeptide.

For certain embodiments of the cell-targeting molecule of the presentinvention, the Shiga toxin A Subunit effector polypeptide is capable ofexhibiting one or more Shiga toxin effector functions in addition todelivery of the CD8+ T-cell epitope-peptide cargo to a MHC class Imolecule of the cell. For certain embodiments of the cell-targetingmolecule of the present invention, the Shiga toxin A Subunit effectorpolypeptide is capable of exhibiting one or more Shiga toxin effectorfunctions in addition to delivery of the CD8+ T-cell epitope-peptidecargo from an early endosomal compartment of a cell in which the Shigatoxin effector polypeptide is present to a MHC class I molecule of thecell.

For certain embodiments of the cell-targeting molecule of the presentinvention, the Shiga toxin A Subunit effector polypeptide is capable ofexhibiting a ribosome inhibition activity with a half-maximal inhibitoryconcentration (IC₅₀) value of 10,000 picomolar or less.

In certain embodiments of the cell-targeting molecule of the presentinvention, the Shiga toxin A Subunit effector polypeptide comprises oneor more mutations relative to a naturally occurring A Subunit of amember of the Shiga toxin family which changes an enzymatic activity ofthe Shiga toxin A Subunit effector polypeptide, the mutation selectedfrom at least one amino acid residue deletion, insertion, orsubstitution. For certain further embodiments, the mutation, relative tothe naturally occurring A Subunit which changes an enzymatic activity ofthe Shiga toxin A Subunit effector polypeptide, reduces or eliminates acytotoxicity exhibited by the Shiga toxin A Subunit effector polypeptidewithout the mutation(s).

Different embodiments of the cell-targeting molecules of the presentinvention are described below with reference to sets of embodimentsnumbered #1-20.

Embodiment Set #1—Cell-Targeting Molecules Comprising a De-ImmunizedShiga Toxin Effector Polypeptide Comprising an Embedded or Inserted,Heterologous, T-Cell Epitope and a Non-Overlapping De-ImmunizedSub-Region

The present invention provides cell-targeting molecules, each comprising(i) a binding region capable of specifically binding an extracellulartarget biomolecule and (ii) a de-immunized. Shiga toxin effectorpolypeptide of Embodiment Set #1 (see e.g. FIG. 1, depictingillustrative examples of four, exemplary embodiments of thecell-targeting molecules of this embodiment set #1). For example,certain embodiments of set #1 is the cell-targeting molecule comprising(i) a binding region capable of specifically binding an extracellulartarget biomolecule and (ii) a de-immunized, Shiga toxin effectorpolypeptide comprising at least one inserted or embedded, heterologousepitope (a) and at least one disrupted, endogenous, B-cell and/or CD4+T-cell epitope region (b), wherein the heterologous epitope does notoverlap with the embedded or inserted, heterologous, T-cell epitope. Forcertain further embodiments, the Shiga toxin effector polypeptide iscapable of exhibiting at least one Shiga toxin effector function, suchas, e.g., directing intracellular routing to the endoplasmic reticulumand/or cytosol of a cell in which the polypeptide is present, inhibitinga ribosome function, enzymatically inactivating a ribosome, causingcytostasis, and/or causing cytotoxicity. In certain further embodiments,the heterologous, T-cell epitope is a CD8+ T-cell epitope, such as,e.g., with regard to a human immune system. For certain furtherembodiments, the heterologous, T-cell epitope is capable of beingpresented by a MHC class I molecule of a cell. In certain furtherembodiments, the cell-targeting molecule of the present invention iscapable of one or more the following: entering a cell, inhibiting aribosome function, causing cytostasis, causing cell death, and/ordelivering the embedded or inserted, heterologous, T-cell epitope to aMHC class I molecule for presentation on a cellular surface. For certainfurther embodiments, the cell-targeting molecule is capable whenintroduced to cells of exhibiting a cytotoxicity comparable or betterthan a reference molecule, such as, e.g., a second cell-targetingmolecule consisting of the cell-targeting molecule except for all of itsShiga toxin effector polypeptide component(s) each comprise a wild-typeShiga toxin A1 fragment.

For certain embodiments of Embodiment Set #1, the cell-targetingmolecule comprises a molecular moiety located carboxy-terminal to thecarboxy-terminus of the Shiga toxin A1 fragment region.

For certain embodiments of Embodiment Set #1, the cell-targetingmolecule of the present invention is capable when introduced to achordate of exhibiting improved in vivo tolerability and/or stabilitycompared to a reference molecule, such as, e.g., a second cell-targetingmolecule consisting of the cell-targeting molecule except for all of itsShiga toxin effector polypeptide component(s) each comprise a wild-typeShiga toxin A1 fragment and/or wild-type Shiga toxin furin-cleavage siteat the carboxy terminus of its A1 fragment region. In certain furtherembodiments, the Shiga toxin effector polypeptide is not cytotoxic andthe molecular moiety is cytotoxic.

In certain embodiments of Embodiment Set #1, the binding region andShiga toxin effector polypeptide are linked together, either directly orindirectly.

In certain embodiments of Embodiment Set #1, the binding regioncomprises a polypeptide comprising an immunoglobulin-type bindingregion. In certain further embodiments, the binding region comprising apolypeptide selected from the group consisting of: an autonomous V_(H)domain, single-domain antibody fragment (sdAb), nanobody, heavychain-antibody domain derived from a camelid (V_(H)H or V_(H) domainfragment), heavy-chain antibody domain derived from a cartilaginous fish(V_(H)H or V_(H) domain fragment), immunoglobulin new antigen receptor(IgNAR), V_(NAR) fragment, single-chain variable fragment (scFv),antibody variable fragment (Fv), complementary determining region 3fragment (CDR3), constrained FR3-CDR3-FR4 polypeptide (FR3-CDR3-FR4), Fdfragment, small modular immunopharmaceutical (SMIP) domain,antigen-binding fragment (Fab), Armadillo repeat polypeptide (ArmRP),fibronectin-derived 10^(th) fibronectin type III domain (10Fn3),tenascin type III domain (TNfn3), ankyrin repeat motif domain,low-density-lipoprotein-receptor-derived A-domain (LDLR-A), lipocalin(anticalin), Kunitz domain. Protein-A-derived Z domain, gamma-Bcrystallin-derived domain, ubiquitin-derived domain (affilin),Sac7d-derived polypeptide (affitin), Fyn-derived SH2 domain,miniprotein, C-type lectin-like domain scaffold, engineered antibodymimic, and any genetically manipulated counterparts of any of theforegoing which retain binding functionality, such as, e.g., wherein therelative orientation or order of the heavy and light chains is reversedor flipped.

For certain embodiments of Embodiment Set #1, the cell-targetingmolecule of the present invention is capable of exhibiting (i) acatalytic activity level comparable to a wild-type Shiga toxin A1fragment or wild-type Shiga toxin effector polypeptide, (ii) a ribosomeinhibition activity with a half-maximal inhibitory concentration (IC₅₀)value of 10,000 picomolar or less, and/or (iii) a significant level ofShiga toxin catalytic activity.

For certain embodiments of Embodiment Set #1, the cell-targetingmolecule of the present invention and/or its Shiga toxin effectorpolypeptide is capable of exhibiting subcellular routing efficiencycomparable to a reference cell-targeting molecule comprising a wild-typeShiga toxin A1 fragment or wild-type Shiga toxin effector polypeptideand/or capable of exhibiting a significant level of intracellularrouting activity to the endoplasmic reticulum and/or cytosol from anendosomal starting location of a cell.

For certain embodiments of Embodiment Set #1, whereby administration ofthe cell-targeting molecule of the present invention to a cellphysically coupled with the extracellular target biomolecule of thecell-targeting molecule's binding region, the cell-targeting molecule iscapable of causing death of the cell. In certain further embodiments,administration of the cell-targeting molecule of the invention to twodifferent populations of cell types which differ with respect to thepresence or level of the extracellular target biomolecule, thecell-targeting molecule is capable of causing cell death to thecell-types physically coupled with an extracellular target biomoleculeof the cytotoxic cell-targeting molecule's binding region at a CD₅₀ atleast three times or less than the CD₅₀ to cell types which are notphysically coupled with an extracellular target biomolecule of thecell-targeting molecule's binding region. For certain embodiments,whereby administration of the cell-targeting molecule of the presentinvention to a first populations of cells whose members are physicallycoupled to extracellular target biomolecules of the cell-targetingmolecule's binding region, and a second population of cells whosemembers are not physically coupled to any extracellular targetbiomolecule of the binding region, the cytotoxic effect of thecell-targeting molecule to members of said first population of cellsrelative to members of said second population of cells is at least3-fold greater. For certain embodiments, whereby administration of thecell-targeting molecule of the present invention to a first populationsof cells whose members are physically coupled to a significant amount ofthe extracellular target biomolecule of the cell-targeting molecule'sbinding region, and a second population of cells whose members are notphysically coupled to a significant amount of any extracellular targetbiomolecule of the binding region, the cytotoxic effect of thecell-targeting molecule to members of said first population of cellsrelative to members of said second population of cells is at least3-fold greater. For certain embodiments, whereby administration of thecell-targeting molecule of the present invention to a first populationof target biomolecule positive cells, and a second population of cellswhose members do not express a significant amount of a targetbiomolecule of the cell-targeting molecule's binding region at acellular surface, the cytotoxic effect of the cell-targeting molecule tomembers of the first population of cells relative to members of thesecond population of cells is at least 3-fold greater.

For certain embodiments of Embodiment Set #1, the cell-targetingmolecule of the present invention is capable when introduced to cells ofexhibiting a cytotoxicity with a half-maximal inhibitory concentration(CD₅₀) value of 300 nM or less and/or capable of exhibiting asignificant level of Shiga toxin cytotoxicity.

For certain embodiments of Embodiment Set #1, the cell-targetingmolecule of the present invention is capable of delivering an embeddedor inserted, heterologous, CD8+ T-cell epitope to a MHC class Ipresentation pathway of a cell for cell-surface presentation of theepitope bound by a MHC class I molecule.

In certain embodiments of Embodiment Set #1, the cell-targeting moleculecomprises a molecular moiety associated with the carboxy-terminus of theShiga toxin effector polypeptide. In certain embodiments, the molecularmoiety comprises or consists of the binding region. In certainembodiments, the molecular moiety comprises at least one amino acid andthe Shiga toxin effector polypeptide is linked to at least one aminoacid residue of the molecular moiety. In certain further embodiments,the molecular moiety and the Shiga toxin effector polypeptide are fusedforming a continuous polypeptide.

In certain embodiments of Embodiment Set #1, the cell-targeting moleculefurther comprises a cytotoxic molecular moiety associated with thecarboxy-terminus of the Shiga toxin effector polypeptide. For certainembodiments, the cytotoxic molecular moiety is a cytotoxic agent, suchas, e.g., a small molecule chemotherapeutic agent, anti-neoplasticagent, cytotoxic antibiotic, alkylating agent, antimetabolite,topoisomerase inhibitor, and/or tubulin inhibitor known to the skilledworker and/or described herein. For certain further embodiments, thecytotoxic molecular moiety is cytotoxic at concentrations of less than10,000, 5,000, 1.000, 500, or 200 pM.

In certain embodiments of Embodiment Set #1, the binding region iscapable of binding to an extracellular target biomolecule selected fromthe group consisting of: CD20, CD22, CD40, CD74, CD79, CD25, CD30,HER2/neu/ErbB2, EGFR, EpCAM, EphB2, prostate-specific membrane antigen.Cripto, CDCP1, endoglin, fibroblast activated protein, Lewis-Y, CD19,CD21, CS1/SLAMF7, CD33, CD52, CD133, CEA, gpA33, mucin, TAG-72,tyrosine-protein kinase transmembrane receptor (ROR1 or NTRKR1),carbonic anhydrase IX, folate binding protein, ganglioside GD2,ganglioside GD3, ganglioside GM2, ganglioside Lewis-Y2, VEGFR, Alpha Vbeta3, AlphaSbeta1, ErbB1/EGFR, Erb3, c-MET, IGF1R, EphA3, TRAIL-R1,TRAIL-R2, RANK, FAP, tenascin, CD64, mesothelin, BRCA1, MART-1/MelanA,gp100, tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, GAGE-1/2, BAGE, RAGE,NY-ESO-1, CDK-4, beta-catenin, MUM-1, caspase-8, KIAA0205, HPVE6,SART-1, PRAME, carcinoembryonic antigen, prostate specific antigen,prostate stem cell antigen, human aspartyl (asparaginyl)beta-hydroxylase, EphA2, HER3/ErbB-3, MUC1, MART-1/MelanA, gp100,tyrosinase associated antigen, HPV-E7, Epstein-Barr virus antigen,Bcr-Abl, alpha-fetoprotein antigen, 17-A1, bladder tumor antigen, SAIL,CD38, CD15, CD23, CD45 (protein tyrosine phosphatase receptor type C),CD53, CD88, CD129, CD183, CD191, CD193, CD244, CD294, CD305, C3AR,FceRIa, galectin-9, IL-1R (interleukin-1 receptor), mrp-14, NKG2Dligand, programmed death-ligand 1 (PD-L1), Siglec-8, Siglec-10, CD49d,CD13, CD44, CD54, CD63, CD69, CD123, TLR4, FceRIa, IgE, CD107a, CD203c,CD14, CD68, CD80, CD86, CD105, CD115, F4/80. ILT-3, galectin-3, CD11a-c,GITRL, MHC class I molecule. MHC class II molecule (optionally complexedwith a peptide), CD284 (TLR4), CD107-Mac3, CD195 (CCR5), HLA-DR,CD16/32, CD282 (TLR2), CD11c, and any immunogenic fragment of any of theforegoing.

In certain embodiments of Embodiment Set #1, the binding region islinked, either directly or indirectly, to the Shiga toxin effectorpolypeptide by at least one covalent bond which is not a disulfide bond.In certain further embodiments, the binding region is fused, eitherdirectly or indirectly, to the carboxy-terminus of the Shiga toxineffector polypeptide to form a single, continuous polypeptide. Incertain further embodiments, the binding region is animmunoglobulin-type binding region.

In certain embodiments of Embodiment Set #1, the disruptedfurin-cleavage motif comprises one or more mutations in the minimal,furin-cleavage site relative to a wild-type Shiga toxin A Subunit. Incertain embodiments, the disrupted furin-cleavage motif is not anamino-terminal truncation of sequences that overlap with part or all ofat least one amino acid residue of the minimal furin-cleavage site. Incertain embodiments, the mutation in the minimal, furin-cleavage site isan amino acid deletion, insertion, and/or substitution of at least oneamino acid residue in the R/Y-x-x-R furin cleavage motif. In certainfurther embodiments, the disrupted furin-cleavage motif comprises atleast one mutation relative to a wild-type Shiga toxin A Subunit, themutation altering at least one amino acid residue in the region nativelypositioned 1) at 248-251 of the A Subunit of Shiga toxin (SEQ ID NOs:1-2 and 4-6), or 2) at 247-250 of the A Subunit of Shiga-like toxin 2(SEQ ID NOs: 3 and 7-18), or the equivalent amino acid sequence positionin any Shiga toxin A Subunit. In certain further embodiments, themutation is an amino acid residue substitution of an arginine residuewith a non-positively charged, amino acid residue.

In certain embodiments of Embodiment Set #1, the cell-targeting moleculeof the present invention is capable when introduced to cells ofexhibiting cytotoxicity comparable to a cytotoxicity of a referencemolecule, such as, e.g., a second cell-targeting molecule consisting ofthe cell-targeting molecule except for all of its Shiga toxin effectorpolypeptide component(s) each comprise a wild-type Shiga toxin A1fragment.

In certain embodiments of Embodiment Set #1, the binding regioncomprises the peptide or polypeptide shown in any one of SEQ ID NOs:39-245.

In certain embodiments of Embodiment Set #1, the cell-targeting moleculeof the present invention comprises or consists essentially of thepolypeptide shown in any one of SEQ ID NOs: 252-255 and 288-748, andoptionally the cell-targeting molecule comprises an amino-terminalmethionine residue.

In certain embodiments of Embodiment Set #1, the binding regionsterically covers the carboxy-terminus of the A1 fragment region.

In certain embodiments of Embodiment Set #1, the molecular moietysterically covers the carboxy-terminus of the A1 fragment region. Incertain further embodiments, the molecular moiety comprises the bindingregion.

In certain embodiments of Embodiment Set #1, the cell-targeting moleculeof the present invention comprises a binding region and/or molecularmoiety located carboxy-terminal to the carboxy-terminus of the Shigatoxin A1 fragment region. In certain further embodiments, the mass ofthe binding region and/or molecular moiety is at least 4.5 kDa, 6, kDa,9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, kDa, 41 kDa, 50 kDa, 100kDa, or greater.

In certain embodiments of Embodiment Set #1, the cell-targeting moleculecomprises a binding region with a mass of at least 4.5 kDa, 6, kDa, 9kDa, 12 kDa, kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein(e.g., cytotoxicity and/or intracellular routing).

In certain embodiments of Embodiment Set #1, the binding region iscomprised within a relatively large, molecular moiety comprising suchas, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein.

In certain embodiments of Embodiment Set #1, the amino-terminus of theShiga toxin effector polypeptide is at and/or proximal to anamino-terminus of a polypeptide component of the cell-targetingmolecule. In certain further embodiments, the binding region is notlocated proximally to the amino-terminus of the cell-targeting moleculerelative to the Shiga toxin effector polypeptide. In certain furtherembodiments, the binding region and Shiga toxin effector polypeptide arephysically arranged or oriented within the cell-targeting molecule suchthat the binding region is not located proximally to the amino-terminusof the Shiga toxin effector polypeptide. In certain further embodiments,the binding region is located within the cell-targeting molecule moreproximal to the carboxy-terminus of the Shiga toxin effector polypeptidethan to the amino-terminus of the Shiga toxin effector polypeptide. Forcertain further embodiments, the cell-targeting molecule of the presentinvention is capable when introduced to cells of exhibiting cytotoxicitythat is greater than that of a third cell-targeting molecule having anamino-terminus and comprising the binding region and the Shiga toxineffector polypeptide which is not positioned at or proximal to theamino-terminus of the third cell-targeting molecule. For certain furtherembodiments, the cell-targeting molecule of the present inventionexhibits cytotoxicity with better optimized, cytotoxic potency, such as,e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity ascompared to the cytotoxicity of the third cell-targeting molecule. Forcertain further embodiments, the cytotoxicity of the cell-targetingmolecule of the present invention to a population of target positivecells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-foldor greater than the cytotoxicity of the third cell-targeting molecule toa second population of target positive cells as assayed by CD₅₀ values.In certain further embodiments, the third cell-targeting molecule doesnot comprise any carboxy-terminal, endoplasmic reticulumretention/retrieval signal motif of the KDEL family.

In certain embodiments of Embodiment Set #1, the amino-terminus of theShiga toxin effector polypeptide is at and/or proximal to anamino-terminus of a polypeptide component of the cell-targetingmolecule. In certain further embodiments, the binding region is notlocated proximally to the amino-terminus of the cell-targeting moleculerelative to the Shiga toxin effector polypeptide. In certain furtherembodiments, the binding region and Shiga toxin effector polypeptide arephysically arranged or oriented within the cell-targeting molecule suchthat the binding region is not located proximally to the amino-terminusof the Shiga toxin effector polypeptide. In certain further embodiments,the binding region is located within the cell-targeting molecule moreproximal to the carboxy-terminus of the Shiga toxin effector polypeptidethan to the amino-terminus of the Shiga toxin effector polypeptide. Forcertain further embodiments, the cell-targeting molecule of the presentinvention is not cytotoxic and is capable when introduced to cells ofexhibiting a greater subcellular routing efficiency from anextracellular space to a subcellular compartment of an endoplasmicreticulum and/or cytosol as compared to the cytotoxicity of a thirdcell-targeting molecule having an amino-terminus and comprising thebinding region and the Shiga toxin effector polypeptide which is notpositioned at or proximal to the amino-terminus of the thirdcell-targeting molecule. In certain further embodiments, the thirdcell-targeting molecule does not comprise any carboxy-terminal,endoplasmic reticulum retention/retrieval signal motif of the KDELfamily.

In certain embodiments of Embodiment Set #1, the amino-terminus of theShiga toxin effector polypeptide is at and/or proximal to anamino-terminus of a polypeptide component of the cell-targetingmolecule. In certain further embodiments, the binding region is notlocated proximally to the amino-terminus of the cell-targeting moleculerelative to the Shiga toxin effector polypeptide. In certain furtherembodiments, the binding region and Shiga toxin effector polypeptide arephysically arranged or oriented within the cell-targeting molecule suchthat the binding region is not located proximally to the amino-terminusof the Shiga toxin effector polypeptide. In certain further embodiments,the binding region is located within the cell-targeting molecule moreproximal to the carboxy-terminus of the Shiga toxin effector polypeptidethan to the amino-terminus of the Shiga toxin effector polypeptide. Forcertain further embodiments, the cell-targeting molecule of the presentinvention exhibits low cytotoxic potency (i.e. is not capable whenintroduced to certain positive target cell types of exhibiting acytotoxicity greater than 1% cell death of a cell population at acell-targeting molecule concentration of 1000 nM, 500 nM, 100 nM, 75 nM,or 50 nM) and is capable when introduced to cells of exhibiting agreater subcellular routing efficiency from an extracellular space to asubcellular compartment of an endoplasmic reticulum and/or cytosol ascompared to the cytotoxicity of a third cell-targeting molecule havingan amino-terminus and comprising the binding region and the Shiga toxineffector polypeptide which is not positioned at or proximal to theamino-terminus of the third cell-targeting molecule. In certain furtherembodiments, the third cell-targeting molecule does not comprise anycarboxy-terminal, endoplasmic reticulum retention/retrieval signal motifof the KDEL family.

In certain embodiments of Embodiment Set #1, the cell-targeting moleculeof the present invention, or a polypeptide component thereof, comprisesa carboxy-terminal, endoplasmic reticulum retention/retrieval signalmotif of a member of the KDEL family. For certain further embodiments,the carboxy-terminal endoplasmic reticulum retention/retrieval signalmotif is selected from the group consisting of: KDEL, HDEF, HDEL, RDEF,RDEL, WDEL, YDEL, HEEF, HEEL, KEEL, REEL, KAEL, KCEL, KFEL, KGEL, KHEL,KLEL, KNEL, KQEL, KREL, KSEL, KVEL, KWEL, KYEL, KEDL, KIEL, DKEL, FDEL,KDEF, KKEL, HADL, HAEL, HIEL, HNEL, HTEL, KTEL, HVEL, NDEL, QDEL, REDL,RNEL, RTDL, RTEL, SDEL, TDEL, SKEL, STEL, and EDEL. In certain furtherembodiments, the cell-targeting molecule of the present invention iscapable when introduced to cells of exhibiting cytotoxicity that isgreater than that of a fourth cell-targeting molecule consisting of thecell-targeting molecule except for it does not comprise anycarboxy-terminal, endoplasmic reticulum retention/retrieval signal motifof the KDEL family. In certain further embodiments, the cell-targetingmolecule of the present invention is capable of exhibiting acytotoxicity with better optimized, cytotoxic potency, such as, e.g.,4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity as compared to areference molecule, such as, e.g., the fourth cell-targeting molecule.In certain further embodiments, the cytotoxicity of the cell-targetingmolecule of the present invention to a population of target positivecells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-foldor greater than the cytotoxicity of the fourth cell-targeting moleculeto a second population of target positive cells as assayed by CD₅₀values.

Embodiment Set #2—Cell-Targeting Molecules Comprising a Carboxy-TerminalEndoplasmic Reticulum Retention/Retrieval Signal Motif and a Shiga ToxinEffector Polypeptide Comprising an Embedded or Inserted, Heterologous,T-Cell Epitope

The present invention provides cell-targeting molecules, each comprising(i) a binding region capable of specifically binding an extracellulartarget biomolecule; (ii) a Shiga toxin effector polypeptide comprisingan inserted or embedded, heterologous, epitope, and (iii) acarboxy-terminal, endoplasmic reticulum retention/retrieval signalmotif. In certain embodiments, the cell-targeting molecule of thepresent invention comprises (a) a binding region capable of specificallybinding at least one extracellular target biomolecule; (b) a Shiga toxineffector polypeptide comprising an embedded or inserted, heterologousepitope; and (c) a carboxy-terminal, endoplasmic reticulumretention/retrieval signal motif of a member of the KDEL family. Forcertain further embodiments, the Shiga toxin effector polypeptide iscapable of exhibiting at least one Shiga toxin effector function, suchas, e.g., directing intracellular routing to the endoplasmic reticulumand/or cytosol of a cell in which the polypeptide is present, inhibitinga ribosome function, enzymatically inactivating a ribosome, causingcytostasis, and/or causing cytotoxicity. In certain further embodiments,the heterologous, T-cell epitope is a CD8+ T-cell epitope, such as,e.g., with regard to a human immune system. For certain furtherembodiments, the heterologous, T-cell epitope is capable of beingpresented by a MHC class I molecule of a cell. In certain furtherembodiments, the cell-targeting molecule of the present invention iscapable of one or more the following: entering a cell, inhibiting aribosome function, causing cytostasis, causing cell death, and/ordelivering the embedded or inserted, heterologous. T-cell epitope to aMHC class I molecule for presentation on a cellular surface.

In certain embodiments of Embodiment Set #2, the carboxy-terminalendoplasmic reticulum retention/retrieval signal motif is selected fromthe group consisting of: KDEL, HDEF, HDEL, RDEF, RDEL. WDEL. YDEL, HEEF,HEEL, KEEL. REEL, KAEL, KCEL, KFEL, KGEL, KHEL, KLEL, KNEL, KQEL, KREL,KSEL, KVEL, KWEL, KYEL, KEDL, KIEL, DKEL, FDEL, KDEF, KKEL, HADL, HAEL,HIEL, HNEL, HTEL, KTEL, HVEL, NDEL, QDEL, REDL, RNEL, RTDL. RTEL, SDEL,TDEL, SKEL, STEL, and EDEL.

In certain embodiments of Embodiment Set #2, the embedded or inserted,heterologous, T-cell epitope disrupts the endogenous, B-cell and/orT-cell epitope region selected from the group of natively positionedShiga toxin A Subunit regions consisting of: (i) 1-15 of any one of SEQID NOs: 1-2 and 4-6; 3-14 of any one of SEQ ID NOs: 3 and 7-18; 26-37 ofany one of SEQ ID NOs: 3 and 7-18; 27-37 of any one of SEQ ID NOs: 1-2and 4-6; 39-48 of any one of SEQ ID NOs: 1-2 and 4-6; 42-48 of any oneof SEQ ID NOs: 3 and 7-18; and 53-66 of any one of SEQ ID NOs: 1-18, orthe equivalent region in a Shiga toxin A Subunit or derivative thereof;(ii) 94-115 of any one of SEQ ID NOs: 1-18; 141-153 of any one of SEQ IDNOs: 1-2 and 4-6; 140-156 of any one of SEQ ID NOs: 3 and 7-18; 179-190of any one of SEQ ID NOs: 1-2 and 4-6; 179-191 of any one of SEQ ID NOs:3 and 7-18; 204 of SEQ ID NO:3; 205 of any one of SEQ ID NOs: 1-2 and4-6; and 210-218 of any one of SEQ ID NOs: 3 and 7-18, or the equivalentregion in a Shiga toxin A Subunit or derivative thereof; and (iii)240-260 of any one of SEQ ID NOs: 3 and 7-18; 243-257 of any one of SEQID NOs: 1-2 and 4-6; 254-268 of any one of SEQ ID NOs: 1-2 and 4-6;262-278 of any one of SEQ ID NOs: 3 and 7-18; 281-297 of any one of SEQID NOs: 3 and 7-18; and 285-293 of any one of SEQ ID NOs: 1-2 and 4-6,or the equivalent region in a Shiga toxin A Subunit or derivativethereof.

In certain further embodiments of Embodiment Set #2, the heterologousepitope is a CD8+ T-cell epitope capable of being presented by a MHCclass I molecule of a cell. In certain further embodiments, theheterologous epitope in is embedded and replaces an equivalent number ofamino acid residues in a wild-type Shiga toxin polypeptide region suchthat the Shiga toxin effector polypeptide has the same total number ofamino acid residues as does the wild-type Shiga toxin polypeptide regionfrom which it is derived. In certain further embodiments of any of theabove, the Shiga toxin effector polypeptide is capable of exhibiting atleast one Shiga toxin effector function selected from: directingintracellular routing to a cytosol of a cell in which the polypeptide ispresent, inhibiting a ribosome function, enzymatically inactivating aribosome, and cytotoxicity.

In certain embodiments of Embodiment Set #2, the cell-targeting moleculeof the present invention is capable when introduced to cells ofexhibiting cytotoxicity that is greater than that of a fifthcell-targeting molecule consisting of the cell-targeting molecule exceptfor it does not comprise any carboxy-terminal, endoplasmic reticulumretention/retrieval signal motif of the KDEL family. In certain furtherembodiments, the cell-targeting molecule of the present invention iscapable of exhibiting a cytotoxicity with better optimized, cytotoxicpotency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greatercytotoxicity as compared to the fifth cell-targeting molecule. Incertain further embodiments, the cytotoxicity of the cell-targetingmolecule of the present invention to a population of target positivecells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-foldor greater than the cytotoxicity of the fifth cell-targeting molecule toa second population of target positive cells as assayed by CD₅₀ values.

For certain embodiments of Embodiment Set #2, the cell-targetingmolecule of the present invention is capable of delivering an embeddedor inserted, heterologous, CD8+ T-cell epitope to a MHC class Ipresentation pathway of a cell for cell-surface presentation of theepitope bound by a MHC class I molecule.

In certain embodiments of Embodiment Set #2, the cell-targeting moleculeis de-immunized due to the embedded or inserted, heterologous, epitope.In certain further embodiments, the cell-targeting molecule is capableof exhibiting less relative antigenicity and/or relative immunogenicityas compared to a reference molecule, such as, e.g., a sixthcell-targeting molecule consisting of the cell-targeting molecule exceptfor it lacks one or more embedded or inserted epitopes present in thecell targeting molecule.

For certain further embodiments of Embodiment Set #2, the cell-targetingmolecule of the present invention is not cytotoxic and is capable whenintroduced to cells of exhibiting a greater subcellular routingefficiency from an extracellular space to a subcellular compartment ofan endoplasmic reticulum and/or cytosol as compared to the cytotoxicityof a reference molecule, such as, e.g., the fifth cell-targetingmolecule.

Embodiment Set #3—Cell-Targeting Molecules Comprising a Shiga ToxinEffector Polypeptide Comprising (i) an Embedded or Inserted,Heterologous, T-Cell Epitope and (ii) a Disrupted, Furin-Cleavage Motif

The present invention provides cell-targeting molecules, each comprising(i) a binding region capable of specifically binding an extracellulartarget biomolecule; (ii) a Shiga toxin effector polypeptide comprisingan inserted or embedded, heterologous, epitope; and (iii) a disruptedfurin-cleavage motif. In certain embodiments, the cell-targetingmolecule of the present invention comprises (i) a binding region capableof specifically binding an extracellular target biomolecule; (ii) aShiga toxin effector polypeptide comprising (a) an inserted or embedded,heterologous, epitope; (b) a Shiga toxin A1 fragment derived regionhaving a carboxy terminus; and (c) a disrupted furin-cleavage motif atthe carboxy-terminus of the A1 fragment region. For certain furtherembodiments, the Shiga toxin effector polypeptide is capable ofexhibiting at least one Shiga toxin effector function, such as, e.g.,directing intracellular routing to the endoplasmic reticulum and/orcytosol of a cell in which the polypeptide is present, inhibiting aribosome function, enzymatically inactivating a ribosome, causingcytostasis, and/or causing cytotoxicity. In certain further embodiments,the heterologous, T-cell epitope is a CD8+ T-cell epitope, such as,e.g., with regard to a human immune system. For certain furtherembodiments, the heterologous, T-cell epitope is capable of beingpresented by a MHC class I molecule of a cell. In certain furtherembodiments, the cell-targeting molecule of the present invention iscapable of one or more the following: entering a cell, inhibiting aribosome function, causing cytostasis, causing cell death, and/ordelivering the embedded or inserted, heterologous. T-cell epitope to aMHC class I molecule for presentation on a cellular surface. For certainfurther embodiments, the cell-targeting molecule is capable whenintroduced to cells of exhibiting a cytotoxicity comparable or betterthan a reference molecule, such as, e.g., a second cell-targetingmolecule consisting of the cell-targeting molecule except for all of itsShiga toxin effector polypeptide components comprise a wild-type Shigatoxin furin-cleavage site at the carboxy terminus of its A1 fragmentregion.

In certain embodiments of Embodiment Set #3, the embedded or inserted,heterologous, T-cell epitope disrupts the endogenous, B-cell and/orT-cell epitope region selected from the group of natively positionedShiga toxin A Subunit regions consisting of (i) 1-15 of any one of SEQID NOs: 1-2 and 4-6; 3-14 of any one of SEQ ID NOs: 3 and 7-18; 26-37 ofany one of SEQ ID NOs: 3 and 7-18; 27-37 of any one of SEQ ID NOs: 1-2and 4-6; 39-48 of any one of SEQ ID NOs: 1-2 and 4-6; 42-48 of any oneof SEQ ID NOs: 3 and 7-18; and 53-66 of any one of SEQ ID NOs: 1-18, orthe equivalent region in a Shiga toxin A Subunit or derivative thereof;(ii) 94-115 of any one of SEQ ID NOs: 1-18; 141-153 of any one of SEQ IDNOs: 1-2 and 4-6; 140-156 of any one of SEQ ID NOs: 3 and 7-18; 179-190of any one of SEQ ID NOs: 1-2 and 4-6; 179-191 of any one of SEQ ID NOs:3 and 7-18; 204 of SEQ ID NO:3; 205 of any one of SEQ ID NOs: 1-2 and4-6; and 210-218 of any one of SEQ ID NOs: 3 and 7-18, or the equivalentregion in a Shiga toxin A Subunit or derivative thereof; and (iii)240-260 of any one of SEQ ID NOs: 3 and 7-18; 243-257 of any one of SEQID NOs: 1-2 and 4-6; 254-268 of any one of SEQ ID NOs: 1-2 and 4-6;262-278 of any one of SEQ ID NOs: 3 and 7-18; 281-297 of any one of SEQID NOs: 3 and 7-18; and 285-293 of any one of SEQ ID NOs: 1-2 and 4-6,or the equivalent region in a Shiga toxin A Subunit or derivativethereof.

In certain embodiments of Embodiment Set #3, the disruptedfurin-cleavage motif comprises one or more mutations, relative to awild-type Shiga toxin A Subunit, the mutation altering at least oneamino acid residue in a region natively positioned at 248-251 of the ASubunit of Shiga toxin (SEQ ID NOs: 1-2 and 4-6), or at 247-250 of the ASubunit of Shiga-like toxin 2 (SEQ ID NOs: 3 and 7-18); or theequivalent region in a Shiga toxin A Subunit or derivative thereof. Incertain further embodiments, the disrupted furin-cleavage motifcomprises one or more mutations, relative to a wild-type Shiga toxin ASubunit, in a minimal furin cleavage site of the furin-cleavage motif.In certain further embodiments the minimal furin cleavage site isrepresented by the consensus amino acid sequence R/Y-x-x-R and/orR-x-x-R.

In certain embodiments of Embodiment Set #3, the cell-targeting moleculecomprises a molecular moiety located carboxy-terminal to thecarboxy-terminus of the Shiga toxin A1 fragment region.

In certain embodiments of Embodiment Set #3, the binding regionsterically covers the carboxy-terminus of the A1 fragment region.

In certain embodiments of Embodiment Set #3, the molecular moietysterically covers the carboxy-terminus of the A1 fragment region. Incertain further embodiments, the molecular moiety comprises the bindingregion.

In certain embodiments of Embodiment Set #3, the cell-targeting moleculeof the present invention comprises a binding region and/or molecularmoiety located carboxy-terminal to the carboxy-terminus of the Shigatoxin A1 fragment region. In certain further embodiments, the mass ofthe binding region and/or molecular moiety is at least 4.5 kDa, 6, kDa,9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, kDa, 41 kDa, 50 kDa, 100kDa, or greater.

In certain embodiments of Embodiment Set #3, the cell-targeting moleculecomprises a binding region with a mass of at least 4.5 kDa, 6, kDa, 9kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein(e.g., cytotoxicity and/or intracellular routing).

In certain embodiments of Embodiment Set #3, the binding region iscomprised within a relatively large, molecular moiety comprising suchas, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein.

In certain embodiments of Embodiment Set #3, the disruptedfurin-cleavage motif comprises an amino acid residue substitution in thefurin-cleavage motif relative to a wild-type Shiga toxin A Subunit. Incertain further embodiments, the substitution of the amino acid residuein the furin-cleavage motif is of an arginine residue with anon-positively charged, amino acid residue selected from the groupconsisting of: alanine, glycine, proline, serine, threonine, aspartate,asparagine, glutamate, glutamine, cysteine, isoleucine, leucine,methionine, valine, phenylalanine, tryptophan, and tyrosine. In certainembodiments, the substitution of the amino acid residue in thefurin-cleavage motif is of an arginine residue with a histidine.

In certain embodiments of Embodiment Set #3, the cell-targeting moleculeis capable when introduced to cells of exhibiting cytotoxicitycomparable to the cytotoxicity of a seventh cell-targeting moleculeconsisting of the cell-targeting molecule except for all of its Shigatoxin effector polypeptide component(s) each comprise a wild-type Shigatoxin A1 fragment and/or wild-type Shiga toxin furin-cleavage site atthe carboxy terminus of its A1 fragment region. In certain furtherembodiments, the cell-targeting molecule of the present invention iscapable when introduced to cells of exhibiting cytotoxicity that is in arange of from 0.1-fold, 0.5-fold, or 0.75-fold to 1.2-fold, 1.5-fold,1.75-fold, 2-fold, 3-fold, 4-fold, or 5-fold of the cytotoxicityexhibited by the seventh cell-targeting molecule.

In certain embodiments of Embodiment Set #3, the cell-targeting moleculeis capable when introduced to a chordate of exhibiting improved, in vivotolerability compared to in vivo tolerability of the seventhcell-targeting molecule.

In certain embodiments of Embodiment Set #3, the cell-targeting moleculeis de-immunized due to the embedded or inserted, heterologous, epitope.In certain further embodiments, the cell-targeting molecule is capableof exhibiting less relative antigenicity and/or relative immunogenicityas compared to a reference molecule, such as, e.g., an eighthcell-targeting molecule consisting of the cell-targeting molecule exceptfor it lacks one or more embedded or inserted epitopes present in thecell targeting molecule.

In certain embodiments of Embodiment Set #3, the cell-targeting moleculeis de-immunized due to the furin-cleavage motif disruption. In certainfurther embodiments, the cell-targeting molecule is capable ofexhibiting less relative antigenicity and/or relative immunogenicity ascompared to a ninth cell-targeting molecule consisting of thecell-targeting molecule except for the furin-cleavage motif is wild-typeand/or all the Shiga toxin effector polypeptide components consist of awild-type Shiga toxin A1 fragment.

Embodiment Set #4—Cell-Targeting Molecules Comprising a Shiga ToxinEffector Polypeptide at or Proximal to an Amino-Terminus and Wherein theShiga Toxin Effector Polypeptide Comprises an Embedded or Inserted,Heterologous, T-Cell Epitope

The present invention provides cell-targeting molecules, each comprising(i) a binding region capable of specifically binding an extracellulartarget biomolecule; (ii) a Shiga toxin effector polypeptide comprisingan inserted or embedded, heterologous, epitope; wherein the Shiga toxineffector polypeptide is at or proximal to an amino-terminus of apolypeptide. In certain embodiments, the cell-targeting molecule of thepresent invention comprises (i) a binding region capable of specificallybinding an extracellular target biomolecule, (ii) a polypeptidecomponent, and (iii) a Shiga toxin effector polypeptide comprising aninserted or embedded, heterologous, epitope; wherein the Shiga toxineffector polypeptide is at or proximal to an amino-terminus of thepolypeptide component of the cell-targeting molecule. In certain furtherembodiments, the binding region and Shiga toxin effector polypeptide arephysically arranged or oriented within the cell-targeting molecule suchthat the binding region is not located proximally to the amino-terminusof the Shiga toxin effector polypeptide. In certain further embodiments,the binding region is located within the cell-targeting molecule moreproximal to the carboxy-terminus of the Shiga toxin effector polypeptidethan to the amino-terminus of the Shiga toxin effector polypeptide. Incertain further embodiments, the binding region is not locatedproximally to an amino-terminus of the cell-targeting molecule relativeto the Shiga toxin effector polypeptide. For certain furtherembodiments, the Shiga toxin effector polypeptide is capable ofexhibiting at least one Shiga toxin effector function, such as, e.g.,directing intracellular routing to the endoplasmic reticulum and/orcytosol of a cell in which the polypeptide is present, inhibiting aribosome function, enzymatically inactivating a ribosome, causingcytostasis, and/or causing cytotoxicity. In certain further embodiments,the heterologous. T-cell epitope is a CD8+ T-cell epitope, such as,e.g., with regard to a human immune system. For certain furtherembodiments, the heterologous, T-cell epitope is capable of beingpresented by a MHC class I molecule of a cell. In certain furtherembodiments, the cell-targeting molecule of the present invention iscapable of one or more the following: entering a cell, inhibiting aribosome function, causing cytostasis, causing cell death, and/ordelivering the embedded or inserted, heterologous, T-cell epitope to aMHC class I molecule for presentation on a cellular surface.

In certain embodiments of Embodiment Set #4, the cell-targeting moleculeof the present invention is capable when introduced to cells ofexhibiting cytotoxicity that is greater than that of a tenthcell-targeting molecule having an amino-terminus and comprising thebinding region and the Shiga toxin effector polypeptide region which isnot positioned at or proximal to the amino-terminus of the tenthcell-targeting molecule. In certain further embodiments, thecell-targeting molecule of the present invention is capable ofexhibiting a cytotoxicity with better optimized, cytotoxic potency, suchas, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity ascompared to the tenth cell-targeting molecule. In certain furtherembodiments, the cytotoxicity of the cell-targeting molecule of thepresent invention to a population of target positive cells is 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater thanthe cytotoxicity of the tenth cell-targeting molecule to a secondpopulation of target positive cells as assayed by CD₅₀ values.

For certain embodiments of Embodiment Set #4, the cell-targetingmolecule of the present invention is capable of delivering an embeddedor inserted, heterologous, CD8+ T-cell epitope to a MHC class Ipresentation pathway of a cell for cell-surface presentation of theepitope bound by a MHC class I molecule.

In certain embodiments of Embodiment Set #4, the cell-targeting moleculeis de-immunized due to the embedded or inserted, heterologous, epitope.In certain further embodiments, the cell-targeting molecule is capableof exhibiting less relative antigenicity and/or relative immunogenicityas compared to a reference molecule, such as, e.g., an eleventhcell-targeting molecule consisting of the cell-targeting molecule exceptfor it lacks one or more embedded or inserted epitopes present in thecell targeting molecule.

For certain further embodiments of Embodiment Set #4, the cell-targetingmolecule of the present invention is not cytotoxic and is capable whenintroduced to cells of exhibiting a greater subcellular routingefficiency from an extracellular space to a subcellular compartment ofan endoplasmic reticulum and/or cytosol as compared to the cytotoxicityof a reference molecule, such as, e.g., the tenth cell-targetingmolecule.

Embodiment Set #5—Cell-Targeting Molecules Comprising a De-ImmunizedShiga Toxin Effector Polypeptide Comprising a Disrupted, Furin-CleavageMotif

The present invention provides cell-targeting molecules, each comprising(i) a binding region capable of specifically binding an extracellulartarget biomolecule and (ii) a de-immunized, Shiga toxin effectorpolypeptide comprising a disrupted furin-cleavage motif. In certainembodiments, the cell-targeting molecule of the present inventioncomprises (i) a binding region capable of specifically binding anextracellular target biomolecule and (ii) a de-immunized, Shiga toxineffector polypeptide comprising (a) a Shiga toxin A1 fragment derivedregion having a carboxy terminus, (b) a disrupted furin-cleavage motifat the carboxy-terminus of the A1 fragment region, and (c) at least onedisrupted, endogenous, B-cell and/or CD4+ T-cell epitope and/or epitoperegion. For certain further embodiments, the Shiga toxin effectorpolypeptide is capable of exhibiting at least one Shiga toxin effectorfunction, such as, e.g., directing intracellular routing to theendoplasmic reticulum and/or cytosol of a cell in which the polypeptideis present, inhibiting a ribosome function, enzymatically inactivating aribosome, causing cytostasis, and/or causing cytotoxicity. In certainfurther embodiments, the cell-targeting molecule of the presentinvention is capable of one or more the following: entering a cell,inhibiting a ribosome function, causing cytostasis, and/or causing celldeath. For certain further embodiments, the cell-targeting molecule iscapable when introduced to cells of exhibiting a cytotoxicity comparableor better than a reference molecule, such as, e.g., a secondcell-targeting molecule consisting of the cell-targeting molecule exceptfor all of its Shiga toxin effector polypeptide components comprise awild-type Shiga toxin furin-cleavage site at the carboxy terminus of itsA1 fragment region.

In certain embodiments of Embodiment Set #5, the Shiga toxin effectorpolypeptide comprises a mutation, relative to a wild-type Shiga toxin ASubunit, in the B-cell and/or T-cell epitope region selected from thegroup of natively positioned Shiga toxin A Subunit regions consisting of1-15 of any one of SEQ ID NOs: 1-2 and 4-6; 3-14 of any one of SEQ IDNOs: 3 and 7-18; 26-37 of any one of SEQ ID NOs: 3 and 7-18; 27-37 ofany one of SEQ ID NOs: 1-2 and 4-6; 39-48 of any one of SEQ ID NOs: 1-2and 4-6; 42-48 of any one of SEQ ID NOs: 3 and 7-18; and 53-66 of anyone of SEQ ID NOs: 1-18; 94-115 of any one of SEQ ID NOs: 1-18; 141-153of any one of SEQ ID NOs: 1-2 and 4-6; 140-156 of any one of SEQ ID NOs:3 and 7-18; 179-190 of any one of SEQ ID NOs: 1-2 and 4-6; 179-191 ofany one of SEQ ID NOs: 3 and 7-18; 204 of SEQ ID NO:3; 205 of any one ofSEQ ID NOs: 1-2 and 4-6; and 210-218 of any one of SEQ ID NOs: 3 and7-18; 240-260 of any one of SEQ ID NOs: 3 and 7-18; 243-257 of any oneof SEQ ID NOs: 1-2 and 4-6; 254-268 of any one of SEQ ID NOs: 1-2 and4-6; 262-278 of any one of SEQ ID NOs: 3 and 7-18; 281-297 of any one ofSEQ ID NOs: 3 and 7-18; and 285-293 of any one of SEQ ID NOs: 1-2 and4-6; or the equivalent region in a Shiga toxin A Subunit or derivativethereof. In certain further embodiments, there is no disruption which isa carboxy-terminal truncation of amino acid residues that overlap withpart or all of at least one disrupted, endogenous, B-cell and/or T-cellepitope and/or epitope region.

In certain embodiments of Embodiment Set #5, the disruptedfurin-cleavage motif comprises one or more mutations, relative to awild-type Shiga toxin A Subunit, the mutation altering at least oneamino acid residue in a region natively positioned at 248-251 of the ASubunit of Shiga toxin (SEQ ID NOs: 1-2 and 4-6), or at 247-250 of the ASubunit of Shiga-like toxin 2 (SEQ ID NOs: 3 and 7-18), or theequivalent region in a Shiga toxin A Subunit or derivative thereof. Incertain further embodiments, the disrupted furin-cleavage motifcomprises one or more mutations, relative to a wild-type Shiga toxin ASubunit, in a minimal furin cleavage site of the furin-cleavage motif.In certain further embodiments the minimal furin cleavage site isrepresented by the consensus amino acid sequence R/Y-x-x-R and/orR-x-x-R.

In certain embodiments of Embodiment Set #5, the cell-targeting moleculecomprises a molecular moiety located carboxy-terminal to thecarboxy-terminus of the Shiga toxin A1 fragment region.

In certain embodiments of Embodiment Set #5, the binding regionsterically covers the carboxy-terminus of the A1 fragment region.

In certain embodiments of Embodiment Set #5, the molecular moietysterically covers the carboxy-terminus of the A1 fragment region. Incertain further embodiments, the molecular moiety comprises the bindingregion.

In certain embodiments of Embodiment Set #5, the cell-targeting moleculeof the present invention comprises a binding region and/or molecularmoiety located carboxy-terminal to the carboxy-terminus of the Shigatoxin A1 fragment region. In certain further embodiments, the mass ofthe binding region and/or molecular moiety is at least 4.5 kDa, 6, kDa,9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, kDa, 41 kDa, 50 kDa, 100kDa, or greater.

In certain embodiments of Embodiment Set #5, the cell-targeting moleculecomprises a binding region with a mass of at least 4.5 kDa, 6, kDa, 9kDa, 12 kDa, kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein(e.g., cytotoxicity and/or intracellular routing).

In certain embodiments of Embodiment Set #5, the binding region iscomprised within a relatively large, molecular moiety comprising suchas, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein.

In certain embodiments of Embodiment Set #5, the disruptedfurin-cleavage motif comprises an amino acid residue substitution in thefurin-cleavage motif relative to a wild-type Shiga toxin A Subunit. Incertain further embodiments, the substitution of the amino acid residuein the furin-cleavage motif is of an arginine residue with anon-positively charged, amino acid residue selected from the groupconsisting of: alanine, glycine, proline, serine, threonine, aspartate,asparagine, glutamate, glutamine, cysteine, isoleucine, leucine,methionine, valine, phenylalanine, tryptophan, and tyrosine. In certainembodiments, the substitution of the amino acid residue in thefurin-cleavage motif is of an arginine residue with a histidine.

In certain embodiments of Embodiment Set #5, the cell-targeting moleculeis capable when introduced to cells of exhibiting cytotoxicitycomparable to the cytotoxicity of a reference molecule, such as, e.g., atwelfth cell-targeting molecule consisting of the cell-targetingmolecule except for all of its Shiga toxin effector polypeptidecomponent(s) each comprise a wild-type Shiga toxin A1 fragment and/orwild-type Shiga toxin furin-cleavage site at the carboxy terminus of itsA1 fragment region. In certain further embodiments, the cell-targetingmolecule of the present invention is capable when introduced to cells ofexhibiting cytotoxicity that is in a range of from 0.1-fold, 0.5-fold,or 0.75-fold to 1.2-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold,or 5-fold of the cytotoxicity exhibited by the twelfth cell-targetingmolecule.

In certain embodiments of Embodiment Set #4, the cell-targeting moleculeis capable when introduced to a chordate of exhibiting improved, in vivotolerability compared to in vivo tolerability of the twelfthcell-targeting molecule.

In certain embodiments of Embodiment Set #4, the cell-targeting moleculeis de-immunized due to the furin-cleavage motif disruption. In certainfurther embodiments, the cell-targeting molecule is capable ofexhibiting less relative antigenicity and/or relative immunogenicity ascompared to a reference cell-targeting molecule consisting of thecell-targeting molecule except for the furin-cleavage motif is wild-typeand/or all the Shiga toxin effector polypeptide components consist of awild-type Shiga toxin A1 fragment, such as, e.g., the twelfthcell-targeting molecule.

In certain embodiments of Embodiment Set #4, the cell-targeting moleculeof the present invention comprises or consists essentially of thepolypeptide shown in any one of SEQ ID NOs: 252-255, 259-271, 274-278and 288-748, and optionally the cell-targeting molecule comprises anamino-terminal methionine residue.

Embodiment Set #6—Cell-Targeting Molecules Comprising a Carboxy-TerminalEndoplasmic Reticulum Retention/Retrieval Signal Motif and aDe-Immunized Shiga Toxin Effector Polypeptide

The present invention provides cell-targeting molecules, each comprising(i) a binding region capable of specifically binding an extracellulartarget biomolecule; (ii) a de-immunized, Shiga toxin effectorpolypeptide, and (iii) a carboxy-terminal, endoplasmic reticulumretention/retrieval signal motif. In certain embodiments, thecell-targeting molecule of the present invention comprises (i) a bindingregion capable of specifically binding an extracellular targetbiomolecule; (ii) a de-immunized. Shiga toxin effector polypeptidecomprising at least one disrupted, endogenous, B-cell and/or CD4+ T-cellepitope and/or epitope region, and (iii) a carboxy-terminal, endoplasmicreticulum retention/retrieval signal motif of a member of the KDELfamily. For certain further embodiments, the Shiga toxin effectorpolypeptide is capable of exhibiting at least one Shiga toxin effectorfunction, such as, e.g., directing intracellular routing to theendoplasmic reticulum and/or cytosol of a cell in which the polypeptideis present, inhibiting a ribosome function, enzymatically inactivating aribosome, causing cytostasis, and/or causing cytotoxicity. In certainfurther embodiments, the cell-targeting molecule of the presentinvention is capable of one or more the following: entering a cell,inhibiting a ribosome function, causing cytostasis, and/or causing celldeath.

In certain embodiments of Embodiment Set #6, the carboxy-terminalendoplasmic reticulum retention/retrieval signal motif is selected fromthe group consisting of: KDEL, HDEF, HDEL, RDEF, RDEL. WDEL. YDEL, HEEF,HEEL, KEEL. REEL, KAEL, KCEL, KFEL, KGEL, KHEL, KLEL, KNEL. KQEL, KREL,KSEL, KVEL, KWEL, KYEL, KEDL, KIEL, DKEL, FDEL. KDEF, KKEL, HADL, HAEL,HIEL, HNEL, HTEL, KTEL, HVEL, NDEL, QDEL, REDL, RNEL, RTDL, RTEL, SDEL,TDEL, SKEL, STEL, and EDEL.

In certain embodiments of Embodiment Set #6, the Shiga toxin effectorpolypeptide comprises a mutation, relative to a wild-type Shiga toxin ASubunit, in the B-cell and/or T-cell epitope region selected from thegroup of natively positioned Shiga toxin A Subunit regions consisting of1-15 of any one of SEQ ID NOs: 1-2 and 4-6; 3-14 of any one of SEQ IDNOs: 3 and 7-18; 26-37 of any one of SEQ ID NOs: 3 and 7-18; 27-37 ofany one of SEQ ID NOs: 1-2 and 4-6; 39-48 of any one of SEQ ID NOs: 1-2and 4-6; 42-48 of any one of SEQ ID NOs: 3 and 7-18; and 53-66 of anyone of SEQ ID NOs: 1-18; 94-115 of any one of SEQ ID NOs: 1-18; 141-153of any one of SEQ ID NOs: 1-2 and 4-6; 140-156 of any one of SEQ ID NOs:3 and 7-18; 179-190 of any one of SEQ ID NOs: 1-2 and 4-6; 179-191 ofany one of SEQ ID NOs: 3 and 7-18; 204 of SEQ ID NO:3; 205 of any one ofSEQ ID NOs: 1-2 and 4-6; and 210-218 of any one of SEQ ID NOs: 3 and7-18; 240-260 of any one of SEQ ID NOs: 3 and 7-18; 243-257 of any oneof SEQ ID NOs: 1-2 and 4-6; 254-268 of any one of SEQ ID NOs: 1-2 and4-6; 262-278 of any one of SEQ ID NOs: 3 and 7-18; 281-297 of any one ofSEQ ID NOs: 3 and 7-18; and 285-293 of any one of SEQ ID NOs: 1-2 and4-6; or the equivalent region in a Shiga toxin A Subunit or derivativethereof. In certain further embodiments, there is no disruption which isa carboxy-terminal truncation of amino acid residues that overlap withpart or all of at least one disrupted, endogenous, B-cell and/or T-cellepitope and/or epitope region.

In certain embodiments of Embodiment Set #6, the cell-targeting moleculeof the present invention is capable when introduced to cells ofexhibiting cytotoxicity that is greater than that of a thirteenthcell-targeting molecule consisting of the cell-targeting molecule exceptfor it does not comprise any carboxy-terminal, endoplasmic reticulumretention/retrieval signal motif of the KDEL family. In certain furtherembodiments, the cell-targeting molecule of the present invention iscapable of exhibiting a cytotoxicity with better optimized, cytotoxicpotency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greatercytotoxicity as compared to the thirteenth cell-targeting molecule. Incertain further embodiments, the cytotoxicity of the cell-targetingmolecule of the present invention to a population of target positivecells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-foldor greater than the cytotoxicity of the thirteenth cell-targetingmolecule to a second population of target positive cells as assayed byCD₅₀ values.

For certain further embodiments of Embodiment Set #6, the cell-targetingmolecule of the present invention is not cytotoxic and is capable whenintroduced to cells of exhibiting a greater subcellular routingefficiency from an extracellular space to a subcellular compartment ofan endoplasmic reticulum and/or cytosol as compared to the cytotoxicityof a reference molecule, such as, e.g., the thirteenth cell-targetingmolecule.

Embodiment Set #7—Cell-Targeting Molecules Comprising a De-ImmunizedShiga Toxin Effector Polypeptide at or Proximal to an Amino-Terminus ofthe Cell Targeting Molecule

The present invention provides cell-targeting molecules, each comprising(i) a binding region capable of specifically binding an extracellulartarget biomolecule. (ii) a de-immunized, Shiga toxin effectorpolypeptide; wherein the Shiga toxin effector polypeptide is at orproximal to an amino-terminus. In certain embodiments, thecell-targeting molecule of the present invention comprises (i) a bindingregion capable of specifically binding an extracellular targetbiomolecule: (ii) polypeptide component; and (iii) a de-immunized, Shigatoxin effector polypeptide comprising at least one disrupted,endogenous, B-cell and/or CD4+ T-cell epitope and/or epitope region;wherein the Shiga toxin effector polypeptide is at or proximal to anamino-terminus of the polypeptide component of the cell-targetingmolecule. In certain further embodiments, the binding region and Shigatoxin effector polypeptide are physically arranged or oriented withinthe cell-targeting molecule such that the binding region is not locatedproximally to the amino-terminus of the Shiga toxin effectorpolypeptide. In certain further embodiments, the binding region islocated within the cell-targeting molecule more proximal to thecarboxy-terminus of the Shiga toxin effector polypeptide than to theamino-terminus of the Shiga toxin effector polypeptide. In certainfurther embodiments, the binding region is not located proximally to anamino-terminus of the cell-targeting molecule relative to the Shigatoxin effector polypeptide. For certain further embodiments, the Shigatoxin effector polypeptide is capable of exhibiting at least one Shigatoxin effector function, such as, e.g., directing intracellular routingto the endoplasmic reticulum and/or cytosol of a cell in which thepolypeptide is present, inhibiting a ribosome function, enzymaticallyinactivating a ribosome, causing cytostasis, and/or causingcytotoxicity. In certain further embodiments, the cell-targetingmolecule of the present invention is capable of one or more thefollowing: entering a cell, inhibiting a ribosome function, causingcytostasis, and/or causing cell death.

In certain embodiments of Embodiment Set #7, the Shiga toxin effectorpolypeptide comprises a mutation, relative to a wild-type Shiga toxin ASubunit, in the B-cell and/or T-cell epitope region selected from thegroup of natively positioned Shiga toxin A Subunit regions consistingof: 11-15 of any one of SEQ ID NOs: 1-2 and 4-6; 3-14 of any one of SEQID NOs: 3 and 7-18; 26-37 of any one of SEQ ID NOs: 3 and 7-18; 27-37 ofany one of SEQ ID NOs: 1-2 and 4-6; 39-48 of any one of SEQ ID NOs: 1-2and 4-6; 42-48 of any one of SEQ ID NOs: 3 and 7-18; and 53-66 of anyone of SEQ ID NOs: 1-18; 94-115 of any one of SEQ ID NOs: 1-18; 141-153of any one of SEQ ID NOs: 1-2 and 4-6; 140-156 of any one of SEQ ID NOs:3 and 7-18; 179-190 of any one of SEQ ID NOs: 1-2 and 4-6; 179-191 ofany one of SEQ ID NOs: 3 and 7-18; 204 of SEQ ID NO:3; 205 of any one ofSEQ ID NOs: 1-2 and 4-6; and 210-218 of any one of SEQ ID NOs: 3 and7-18; 240-260 of any one of SEQ ID NOs: 3 and 7-18; 243-257 of any oneof SEQ ID NOs: 1-2 and 4-6; 254-268 of any one of SEQ ID NOs: 1-2 and4-6; 262-278 of any one of SEQ ID NOs: 3 and 7-18; 281-297 of any one ofSEQ ID NOs: 3 and 7-18; and 285-293 of any one of SEQ ID NOs: 1-2 and4-6; or the equivalent region in a Shiga toxin A Subunit or derivativethereof. In certain further embodiments, there is no disruption which isa carboxy-terminal truncation of amino acid residues that overlap withpart or all of at least one disrupted, endogenous, B-cell and/or T-cellepitope and/or epitope region.

In certain embodiments of Embodiment Set #7, the cell-targeting moleculeof the present invention is capable when introduced to cells ofexhibiting cytotoxicity that is greater than that of a fourteenthcell-targeting molecule having an amino-terminus and comprising thebinding region and the Shiga toxin effector polypeptide region which isnot positioned at or proximal to the amino-terminus of the fourteenthcell-targeting molecule. In certain further embodiments, thecell-targeting molecule of the present invention is capable ofexhibiting a cytotoxicity with better optimized, cytotoxic potency, suchas, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity ascompared to the fourteenth cell-targeting molecule. In certain furtherembodiments, the cytotoxicity of the cell-targeting molecule of thepresent invention to a population of target positive cells is 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater thanthe cytotoxicity of the fourteenth cell-targeting molecule to a secondpopulation of target positive cells as assayed by CD₅₀ values.

For certain further embodiments of Embodiment Set #7, the cell-targetingmolecule of the present invention is not cylotoxic and is capable whenintroduced to cells of exhibiting a greater subcellular routingefficiency from an extracellular space to a subcellular compartment ofan endoplasmic reticulum and/or cytosol as compared to the cytotoxicityof a reference molecule, such as, e.g., the fourteenth cell-targetingmolecule.

In certain embodiments of Embodiment Set #7, the cell-targeting moleculeof the present invention comprises or consists essentially of thepolypeptide shown in any one of SEQ ID NOs: 252-255, 259-271, 274-278and 288-748, and optionally the cell-targeting molecule comprises anamino-terminal methionine residue.

Embodiment Set #8—Cell-Targeting Molecules Comprising a Carboxy-TerminalEndoplasmic Reticulum Retention/Retrieval Signal Motif and a Shiga ToxinEffector Polypeptide Comprising a Disrupted, Furin-Cleavage Motif

The present invention provides cell-targeting molecules, each comprising(i) a binding region capable of specifically binding an extracellulartarget biomolecule; (ii) a Shiga toxin effector polypeptide comprising adisrupted furin-cleavage motif; and (iii) a carboxy-terminal endoplasmicreticulum retention/retrieval signal motif. The present inventionprovides cell-targeting molecules, each comprising (i) a binding regioncapable of specifically binding an extracellular target biomolecule;(ii) a Shiga toxin effector polypeptide comprising a disruptedfurin-cleavage motif; and (iii) a carboxy-terminal, endoplasmicreticulum retention/retrieval signal motif of a member of the KDELfamily. For certain further embodiments, the Shiga toxin effectorpolypeptide is capable of exhibiting at least one Shiga toxin effectorfunction, such as, e.g., directing intracellular routing to theendoplasmic reticulum and/or cytosol of a cell in which the polypeptideis present, inhibiting a ribosome function, enzymatically inactivating aribosome, causing cytostasis, and/or causing cytotoxicity. In certainfurther embodiments, the cell-targeting molecule of the presentinvention is capable of one or more the following: entering a cell,inhibiting a ribosome function, causing cytostasis, and/or causing celldeath. For certain further embodiments, the cell-targeting molecule iscapable when introduced to cells of exhibiting a cytotoxicity comparableor better than a reference molecule, such as, e.g., a secondcell-targeting molecule consisting of the cell-targeting molecule exceptfor all of its Shiga toxin effector polypeptide components comprise awild-type Shiga toxin furin-cleavage site at the carboxy terminus of itsA1 fragment region.

In certain embodiments of Embodiment Set #8, the disruptedfurin-cleavage motif comprises one or more mutations, relative to awild-type Shiga toxin A Subunit, the mutation altering at least oneamino acid residue in a region natively positioned at 248-251 of the ASubunit of Shiga toxin (SEQ ID NOs: 1-2 and 4-6), or at 247-250 of the ASubunit of Shiga-like toxin 2 (SEQ ID NOs: 3 and 7-18), or theequivalent region in a Shiga toxin A Subunit or derivative thereof. Incertain further embodiments, the disrupted furin-cleavage motifcomprises one or more mutations, relative to a wild-type Shiga toxin ASubunit, in a minimal furin cleavage site of the furin-cleavage motif.In certain further embodiments the minimal furin cleavage site isrepresented by the consensus amino acid sequence R/Y-x-x-R and/orR-x-x-R

In certain embodiments of Embodiment Set #8, the cell-targeting moleculecomprises a molecular moiety located carboxy-terminal to thecarboxy-terminus of the Shiga toxin A1 fragment region.

In certain embodiments of Embodiment Set #8, the binding regionsterically covers the carboxy-terminus of the A1 fragment region.

In certain embodiments of Embodiment Set #8, the molecular moietysterically covers the carboxy-terminus of the A1 fragment region. Incertain further embodiments, the molecular moiety comprises the bindingregion.

In certain embodiments of Embodiment Set #8, the cell-targeting moleculeof the present invention comprises a binding region and/or molecularmoiety located carboxy-terminal to the carboxy-terminus of the Shigatoxin A1 fragment region. In certain further embodiments, the mass ofthe binding region and/or molecular moiety is at least 4.5 kDa, 6, kDa,9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, kDa, 41 kDa, 50 kDa, 100kDa, or greater.

In certain embodiments of Embodiment Set #8, the cell-targeting moleculecomprises a binding region with a mass of at least 4.5 kDa, 6, kDa, 9kDa, 12 kDa, kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein(e.g., cytotoxicity and/or intracellular routing).

In certain embodiments of Embodiment Set #8, the binding region iscomprised within a relatively large, molecular moiety comprising suchas, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein.

In certain embodiments of Embodiment Set #8, the disruptedfurin-cleavage motif comprises an amino acid residue substitution in thefurin-cleavage motif relative to a wild-type Shiga toxin A Subunit. Incertain further embodiments, the substitution of the amino acid residuein the furin-cleavage motif is of an arginine residue with anon-positively charged, amino acid residue selected from the groupconsisting of: alanine, glycine, proline, serine, threonine, aspartate,asparagine, glutamate, glutamine, cysteine, isoleucine, leucine,methionine, valine, phenylalanine, tryptophan, and tyrosine. In certainembodiments, the substitution of the amino acid residue in thefurin-cleavage motif is of an arginine residue with a histidine.

In certain embodiments of Embodiment Set #8, the cell-targeting moleculeof the present invention is capable when introduced to cells ofexhibiting cytotoxicity that is greater than that of a fifteenthcell-targeting molecule consisting of the cell-targeting molecule exceptfor it does not comprise any carboxy-terminal, endoplasmic reticulumretention/retrieval signal motif of the KDEL family. In certain furtherembodiments, the cell-targeting molecule of the present invention iscapable of exhibiting a cytotoxicity with better optimized, cytotoxicpotency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greatercytotoxicity as compared to the fifteenth cell-targeting molecule. Incertain further embodiments, the cytotoxicity of the cell-targetingmolecule of the present invention to a population of target positivecells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-foldor greater than the cytotoxicity of the fifteenth cell-targetingmolecule to a second population of target positive cells as assayed byCD₅₀ values.

In certain embodiments of Embodiment Set #8, the cell-targeting moleculeis capable when introduced to a chordate of exhibiting improved, in vivotolerability compared to in vivo tolerability of a sixteenthcell-targeting molecule consisting of the cell-targeting molecule exceptfor all of its Shiga toxin effector polypeptide component(s) eachcomprise a wild-type Shiga toxin A1 fragment and/or wild-type Shigatoxin furin-cleavage site at the carboxy terminus of its A1 fragmentregion.

In certain embodiments of Embodiment Set #8, the cell-targeting moleculeis de-immunized due to the furin-cleavage motif disruption. In certainfurther embodiments, the cell-targeting molecule is capable ofexhibiting less relative antigenicity and/or relative immunogenicity ascompared to a reference cell-targeting molecule consisting of thecell-targeting molecule except for the furin-cleavage motif is wild-typeand/or all the Shiga toxin effector polypeptide components consist of awild-type Shiga toxin A1 fragment, such as, e.g., the sixteenthcell-targeting molecule.

For certain further embodiments of Embodiment Set #8, the cell-targetingmolecule of the present invention is not cytotoxic and is capable whenintroduced to cells of exhibiting a greater subcellular routingefficiency from an extracellular space to a subcellular compartment ofan endoplasmic reticulum and/or cytosol as compared to the cytotoxicityof a reference molecule, such as, e.g., the fifteenth cell-targetingmolecule.

Embodiment Set #9—Cell-Targeting Molecules Comprising a Furin-CleavageResistant Shiga Toxin Effector Polypeptide at or Proximal to anAmino-Terminus of the Cell Targeting Molecule

The present invention provides cell-targeting molecules, each comprising(i) a binding region capable of specifically binding an extracellulartarget biomolecule and (ii) a Shiga toxin effector polypeptidecomprising a disrupted furin-cleavage motif at the carboxy-terminus ofits Shiga toxin A1 fragment region; wherein the amino-terminus of theShiga toxin effector polypeptide is at and/or proximal to anamino-terminus of a polypeptide component of the cell-targetingmolecule. In certain embodiments, the cell-targeting molecule of thepresent invention comprises (i) a binding region capable of specificallybinding an extracellular target biomolecule, (ii) a Shiga toxin effectorpolypeptide having an amino-terminus and a Shiga toxin A1 fragmentderived region having a carboxy terminus, and (iii) a disruptedfurin-cleavage motif at the carboxy-terminus of the A1 fragment region;wherein the binding region is not located proximally to theamino-terminus of the cell-targeting molecule relative to the Shigatoxin effector polypeptide. In certain further embodiments, the bindingregion and Shiga toxin effector polypeptide are physically arranged ororiented within the cell-targeting molecule such that the binding regionis not located proximally to the amino-terminus of the Shiga toxineffector polypeptide. In certain further embodiments, the binding regionis located within the cell-targeting molecule more proximal to thecarboxy-terminus of the Shiga toxin effector polypeptide than to theamino-terminus of the Shiga toxin effector polypeptide. In certainfurther embodiments, the binding region is not located proximally to anamino-terminus of the cell-targeting molecule relative to the Shigatoxin effector polypeptide. For certain further embodiments, the Shigatoxin effector polypeptide is capable of exhibiting at least one Shigatoxin effector function, such as, e.g., directing intracellular routingto the endoplasmic reticulum and/or cytosol of a cell in which thepolypeptide is present, inhibiting a ribosome function, enzymaticallyinactivating a ribosome, causing cytostasis, and/or causingcytotoxicity. In certain further embodiments, the cell-targetingmolecule of the present invention is capable of one or more thefollowing: entering a cell, inhibiting a ribosome function, causingcytostasis, and/or causing cell death. For certain further embodiments,the cell-targeting molecule is capable when introduced to cells ofexhibiting a cytotoxicity comparable or better than a referencemolecule, such as, e.g., a seventeenth cell-targeting moleculeconsisting of the cell-targeting molecule except for all of its Shigatoxin effector polypeptide components comprise a wild-type Shiga toxinfurin-cleavage site at the carboxy terminus of its A1 fragment region.

In certain embodiments of Embodiment Set #9, the disruptedfurin-cleavage motif comprises one or more mutations, relative to awild-type Shiga toxin A Subunit, the mutation altering at least oneamino acid residue in a region natively positioned at 248-251 of the ASubunit of Shiga toxin (SEQ ID NOs: 1-2 and 4-6), or at 247-250 of the ASubunit of Shiga-like toxin 2 (SEQ ID NOs: 3 and 7-18), or theequivalent region in a Shiga toxin A Subunit or derivative thereof. Incertain further embodiments, the disrupted furin-cleavage motifcomprises one or more mutations, relative to a wild-type Shiga toxin ASubunit, in a minimal furin cleavage site of the furin-cleavage motif.In certain further embodiments the minimal furin cleavage site isrepresented by the consensus amino acid sequence R/Y-x-x-R and/orR-x-x-R.

In certain embodiments of Embodiment Set #9, the cell-targeting moleculecomprises a molecular moiety located carboxy-terminal to thecarboxy-terminus of the Shiga toxin A1 fragment region.

In certain embodiments of Embodiment Set #9, the binding regionsterically covers the carboxy-terminus of the A1 fragment region.

In certain embodiments of Embodiment Set #9, the molecular moietysterically covers the carboxy-terminus of the A1 fragment region. Incertain further embodiments, the molecular moiety comprises the bindingregion.

In certain embodiments of Embodiment Set #9, the cell-targeting moleculeof the present invention comprises a binding region and/or molecularmoiety located carboxy-terminal to the carboxy-terminus of the Shigatoxin A1 fragment region. In certain further embodiments, the mass ofthe binding region and/or molecular moiety is at least 4.5 kDa, 6, kDa,9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, kDa, 41 kDa, 50 kDa, 100kDa, or greater.

In certain embodiments of Embodiment Set #9, the cell-targeting moleculecomprises a binding region with a mass of at least 4.5 kDa, 6, kDa, 9kDa, 12 kDa, kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein(e.g., cytotoxicity and/or intracellular routing).

In certain embodiments of Embodiment Set #9, the binding region iscomprised within a relatively large, molecular moiety comprising suchas, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein.

In certain embodiments of Embodiment Set #9, the disruptedfurin-cleavage motif comprises an amino acid residue substitution in thefurin-cleavage motif relative to a wild-type Shiga toxin A Subunit. Incertain further embodiments, the substitution of the amino acid residuein the furin-cleavage motif is of an arginine residue with anon-positively charged, amino acid residue selected from the groupconsisting of alanine, glycine, proline, serine, threonine, aspartate,asparagine, glutamate, glutamine, cysteine, isoleucine, leucine,methionine, valine, phenylalanine, tryptophan, and tyrosine. In certainembodiments, the substitution of the amino acid residue in thefurin-cleavage motif is of an arginine residue with a histidine.

In certain embodiments of Embodiment Set #9, the cell-targeting moleculeof the present invention is capable when introduced to cells ofexhibiting cytotoxicity that is greater than that of an eighteenthcell-targeting molecule having an amino-terminus and comprising thebinding region and the Shiga toxin effector polypeptide region which isnot positioned at or proximal to the amino-terminus of the eighteenthcell-targeting molecule. In certain further embodiments, thecell-targeting molecule of the present invention is capable ofexhibiting a cytotoxicity with better optimized, cytotoxic potency, suchas, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity ascompared to the eighteenth cell-targeting molecule. In certain furtherembodiments, the cytotoxicity of the cell-targeting molecule of thepresent invention to a population of target positive cells is 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater thanthe cytotoxicity of the eighteenth cell-targeting molecule to a secondpopulation of target positive cells as assayed by CD₅₀ values.

In certain embodiments of Embodiment Set #9, the cell-targeting moleculeis capable when introduced to a chordate of exhibiting improved, in vivotolerability compared to in vivo tolerability of a nineteenthcell-targeting molecule consisting of the cell-targeting molecule exceptfor all of its Shiga toxin effector polypeptide component(s) eachcomprise a wild-type Shiga toxin A1 fragment and/or wild-type Shigatoxin furin-cleavage site at the carboxy terminus of its A1 fragmentregion.

In certain embodiments of Embodiment Set #9, the cell-targeting moleculeis de-immunized due to the furin-cleavage motif disruption. In certainfurther embodiments, the cell-targeting molecule is capable ofexhibiting less relative antigenicity and/or relative immunogenicity ascompared to a reference cell-targeting molecule consisting of thecell-targeting molecule except for the furin-cleavage motif is wild-typeand/or all the Shiga toxin effector polypeptide components consist of awild-type Shiga toxin A1 fragment, such as, e.g., the nineteenthcell-targeting molecule.

For certain further embodiments of Embodiment Set #9, the cell-targetingmolecule of the present invention is not cytotoxic and is capable whenintroduced to cells of exhibiting a greater subcellular routingefficiency from an extracellular space to a subcellular compartment ofan endoplasmic reticulum and/or cytosol as compared to the cytotoxicityof a reference molecule, such as, e.g., the nineteenth cell-targetingmolecule.

In certain embodiments of Embodiment Set #9, the cell-targeting moleculeof the present invention comprises or consists essentially of thepolypeptide shown in any one of SEQ ID NOs: 252-255, 259-271, 274-278and 288-748, and optionally the cell-targeting molecule comprises anamino-terminal methionine residue.

Embodiment Set #10—Cell-Targeting Molecules Comprising aCarboxy-Terminal Endoplasmic Reticulum Retention/Retrieval Signal Motifand Shiga Toxin Effector Polypeptide at or Proximal to an Amino-Terminusof the Cell Targeting Molecule

The present invention provides cell-targeting molecules, each comprising(i) a binding region capable of specifically binding an extracellulartarget biomolecule, (ii) a carboxy-terminal, endoplasmic reticulumretention/retrieval signal motif, and (iii) a Shiga toxin effectorpolypeptide; wherein the amino-terminus of the Shiga toxin effectorpolypeptide is at and/or proximal to an amino-terminus of a polypeptidecomponent of the cell-targeting molecule. In certain embodiments, thecell-targeting molecule of the present invention comprises a (i) bindingregion capable of specifically binding an extracellular targetbiomolecule, (ii) a carboxy-terminal, endoplasmic reticulumretention/retrieval signal motif of a member of the KDEL family, (iii) apolypeptide component, and (iv) a Shiga toxin effector polypeptide;wherein the amino-terminus of the Shiga toxin effector polypeptide is atand/or proximal to an amino-terminus of a polypeptide component of thecell-targeting molecule. In certain further embodiments, the bindingregion and Shiga toxin effector polypeptide are physically arranged ororiented within the cell-targeting molecule such that the binding regionis not located proximally to the amino-terminus of the Shiga toxineffector polypeptide. In certain further embodiments, the binding regionis located within the cell-targeting molecule more proximal to thecarboxy-terminus of the Shiga toxin effector polypeptide than to theamino-terminus of the Shiga toxin effector polypeptide. In certainfurther embodiments, the binding region is not located proximally to anamino-terminus of the cell-targeting molecule relative to the Shigatoxin effector polypeptide.

For certain further embodiments, the Shiga toxin effector polypeptide iscapable of exhibiting at least one Shiga toxin effector function, suchas, e.g., directing intracellular routing to the endoplasmic reticulumand/or cytosol of a cell in which the polypeptide is present, inhibitinga ribosome function, enzymatically inactivating a ribosome, causingcytostasis, and/or causing cytotoxicity. In certain further embodiments,the cell-targeting molecule of the present invention is capable of oneor more the following: entering a cell, inhibiting a ribosome function,causing cytostasis, and/or causing cell death.

In certain embodiments of Embodiment Set #10, the carboxy-terminalendoplasmic reticulum retention/retrieval signal motif is selected fromthe group consisting of: KDEL, HDEF, HDEL, RDEF, RDEL. WDEL. YDEL, HEEF,HEEL, KEEL, REEL. KAEL, KCEL, KFEL, KGEL KHEL, KLEL, KNEL, KQEL, KREL,KSEL, KVEL, KWEL, KYEL, KEDL, KIEL, DKEL, FDEL. KDEF, KKEL, HADL HAEL,HIEL, HNEL, HTEL, KTEL, HVEL, NDEL. QDEL, REDL, RNEL, RTDL. RTEL, SDEL,TDEL, SKEL, STEL, and EDEL.

In certain embodiments of Embodiment Set #10, the cell-targetingmolecule of the present invention is capable when introduced to cells ofexhibiting cytotoxicity that is greater than that of a twentiethcell-targeting molecule having an amino-terminus and comprising thebinding region and the Shiga toxin effector polypeptide region which isnot positioned at or proximal to the amino-terminus of the twentiethcell-targeting molecule and/or greater than that of a twenty-firstcell-targeting molecule consisting of the cell-targeting molecule exceptfor it does not comprise any carboxy-terminal, endoplasmic reticulumretention/retrieval signal motif of the KDEL family. In certain furtherembodiments, the twentieth cell-targeting molecule does not comprise anycarboxy-terminal, endoplasmic reticulum retention/retrieval signal motifof the KDEL family. In certain further embodiments, the cell-targetingmolecule of the present invention is capable of exhibiting acytotoxicity with better optimized, cytotoxic potency, such as, e.g.,4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity as compared to areference molecule, such as, e.g., the twentieth and/or twenty-firstcell-targeting molecules. In certain further embodiments, thecytotoxicity of the cell-targeting molecule of the present invention toa population of target positive cells is 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold or greater than the cytotoxicity of thetwentieth and/or twenty-first cell-targeting molecules to a secondpopulation of target positive cells as assayed by CD₅₀ values.

For certain further embodiments of Embodiment Set #10, thecell-targeting molecule of the present invention is not cytotoxic and iscapable when introduced to cells of exhibiting a greater subcellularrouting efficiency from an extracellular space to a subcellularcompartment of an endoplasmic reticulum and/or cytosol as compared tothe cytotoxicity of a reference molecule, such as, e.g., the twentiethand/or twenty-first cell-targeting molecules.

Multivalent Cell-Targeting Molecules of the Present Invention

In certain embodiments, the cell-targeting molecule of the presentinvention is multivalent. In certain embodiments, the multivalentcell-targeting molecule of the present invention comprises two or morebinding regions, wherein each binding region is capable of specificallybinding an extracellular part of the same extracellular targetbiomolecule. For certain further embodiments, upon administration of themultivalent cell-targeting molecule to a population of cells physicallycoupled with target biomolecule, which have the extracellular part boundby two or more binding regions of the multivalent cell-targetingmolecule, results in a cytotoxic effect which is greater than acytotoxic effect resulting from administration of an equivalent amount,mass, or molarity of a monovalent target-binding molecule component ofthe multivalent cell-targeting molecule to a population of the sametarget-positive cells under same conditions by a factor of 2, 2.5, 3, 4,5, 7.5, 10, or greater than the fold-change in target-binding betweenthe monovalent target-binding molecule component and the multivalentcell-targeting molecule as measured by dissociation constant (K_(D)).

Embodiment Set #11—Multivalent Cell-Targeting Molecules Comprising aDe-Immunized Shiga Toxin Effector Polypeptide Comprising an Embedded orInserted Heterologous, T-Cell Epitope and a Non-Overlapping De-ImmunizedSub-Region

The present invention provides multivalent cell-targeting moleculescomprising (i) two or more binding regions, each capable of specificallybinding an extracellular part of the same target biomolecule, and (ii)at least one de-immunized. Shiga toxin effector polypeptide. Forexample, certain Embodiments of Set #11 is the multivalentcell-targeting molecule comprising (i) two or more binding regions eachof which is capable of specifically binding the same extracellulartarget biomolecule and (ii) at least one, de-immunized, Shiga toxineffector polypeptide comprising at least one inserted or embedded,heterologous epitope (a) and at least one disrupted, endogenous, B-celland/or CD4+ T-cell epitope region (b), wherein the heterologous epitopedoes not overlap with the embedded or inserted, heterologous, T-cellepitope. For certain further embodiments, the at least one,de-immunized. Shiga toxin effector polypeptide is capable of exhibitingat least one Shiga toxin effector function, such as, e.g., directingintracellular routing to the endoplasmic reticulum and/or cytosol of acell in which the polypeptide is present, inhibiting a ribosomefunction, enzymatically inactivating a ribosome, causing cytostasis,and/or causing cytotoxicity. For certain further embodiments, uponadministration of the multivalent cell-targeting molecule to a pluralityof cells physically coupled with an extracellular target biomolecule ofthe two or more binding regions, which have the extracellular part boundby two or more binding regions, at a concentration of multivalentcell-targeting molecule equivalent to five percent, ten percent, twentypercent, thirty-five percent, fifty percent, sixty-five percent,seventy-five percent, and/or eighty percent cell-surface occupancy, themajority of the multivalent cell-targeting molecule internalizes intothe plurality of cells in about fifteen hours, ten hours, five hours,four hours, three hours, two hours, one hour, thirty minutes, or less ata physiological temperature appropriate for the cell and/or at about 37degrees Celsius. In certain further embodiments, the heterologous,T-cell epitope is a CD8+ T-cell epitope, such as, e.g., with regard to ahuman immune system. For certain further embodiments, the heterologous,T-cell epitope is capable of being presented by a MHC class I moleculeof a cell. For certain further embodiments, the multivalentcell-targeting molecule of the present invention is capable of one ormore the following: entering a cell, inhibiting a ribosome function,causing cytostasis, causing caspase activation, causing cell death,and/or delivering the embedded or inserted, heterologous, T-cell epitopeto a MHC class I molecule for presentation on a cellular surface. Forcertain further embodiments, the multivalent cell-targeting molecule iscapable when introduced to cells of exhibiting a cytotoxicity comparableor greater than the cytotoxicity of a reference molecule introduced tothe same type of cells. Non-limiting examples of references moleculesinclude a second cell-targeting molecule, such as, e.g., (1) amonovalent second cell-targeting molecule comprising only one of the twoor more binding regions of the multivalent cell-targeting molecule ofinterest and one or more of the same Shiga toxin effector polypeptidecomponent(s) of the multivalent cell-targeting molecule, and/or (2) amultivalent third cell-targeting molecule consisting of the multivalentcell-targeting molecule except for all of its Shiga toxin effectorpolypeptide component(s) each comprises a wild-type Shiga toxin A1fragment.

Each of the at least one, de-immunized, Shiga toxin effectorpolypeptides has an amino terminus, whether with regard to a polypeptideregional boundary and/or a physical polypeptide terminus.

In certain embodiments, the at least one, de-immunized. Shiga toxineffector polypeptide comprises a Shiga toxin A1 fragment region and/orShiga toxin A1 fragment derived region having a carboxy-terminus. Incertain embodiments, the at least one, de-immunized, Shiga toxineffector polypeptide comprises a Shiga toxin A1 fragment region and/orShiga toxin A1 fragment derived region having a carboxy-terminus. Incertain further embodiments, all of the Shiga toxin effector polypeptidecomponents of the multivalent cell-targeting molecule each comprises aShiga toxin A1 fragment region and/or Shiga toxin A1 fragment derivedregion having a carboxy-terminus.

In certain embodiments of Embodiment Set #11, the multivalentcell-targeting molecule comprises a molecular moiety locatedcarboxy-terminal to the carboxy-terminus of the Shiga toxin A1 fragmentregion and/or Shiga toxin A1 fragment derived region of at least one,de-immunized, Shiga toxin effector polypeptide.

For certain embodiments of Embodiment Set #11, the multivalentcell-targeting molecule of the present invention is capable whenintroduced to a chordate of exhibiting improved in vivo tolerabilityand/or stability compared to a reference molecule, such as, e.g., afourth cell-targeting molecule consisting of the multivalentcell-targeting molecule except for at least one of the fourthcell-targeting molecule's Shiga toxin effector polypeptide component(s)comprises a wild-type Shiga toxin A1 fragment and/or wild-type Shigatoxin furin-cleavage site at the carboxy terminus of its A1 fragmentregion or Shiga toxin A1 fragment derived region. For certain furtherembodiments, (1) all the de-immunized, Shiga toxin effectorpolypeptide(s) located amino-terminal to a molecular moiety is notcytotoxic and (2) the molecular moiety is cytotoxic.

In certain embodiments of Embodiment Set #11, at least one of the two ormore binding regions and the at least one, de-immunized, Shiga toxineffector polypeptide are linked together, either directly or indirectly,such as, e.g., being fused to form a continuous polypeptide (see e.g.FIG. 1). In certain further embodiments, all of the two or more bindingregions are each linked, either directly or indirectly, to ade-immunized Shiga toxin effector polypeptide component of themultivalent cell-targeting molecule (see e.g. FIG. 1). In certainfurther embodiments, all the two or more binding regions and all thede-immunized, Shiga toxin effector polypeptides are linked together,either directly or indirectly, such as, e.g., being fused to form acontinuous polypeptide (see e.g. FIG. 1).

In certain embodiments of Embodiment Set #11, at least one of the two ormore binding regions comprises a polypeptide comprising animmunoglobulin-type binding region. In certain further embodiments, atleast one of the two or more binding regions comprises a polypeptideselected from the group consisting of: an autonomous V_(H) domain,single-domain antibody fragment (sdAb), nanobody, heavy chain-antibodydomain derived from a camelid (V₁₁H or V₁₁ domain fragment), heavy-chainantibody domain derived from a cartilaginous fish (V_(H)H or V_(H)domain fragment), immunoglobulin new antigen receptor (IgNAR), V_(NAR)fragment, single-chain variable fragment (scFv), antibody variablefragment (Fv), complementary determining region 3 fragment (CDR3),constrained FR3-CDR3-FR4 polypeptide (FR3-CDR3-FR4), Fd fragment, smallmodular immunopharmaceutical (SMIP) domain, antigen-binding fragment(Fab), Armadillo repeat polypeptide (ArmRP), fibronectin-derived 10^(th)fibronectin type III domain (10Fn3), tenascin type III domain (TNfn3),ankyrin repeat motif domain, low-density-lipoprotein-receptor-derivedA-domain (LDLR-A), lipocalin (anticalin), Kunitz domain,Protein-A-derived Z domain, gamma-B crystallin-derived domain,ubiquitin-derived domain, Sac7d-derived polypeptide (affitin),Fyn-derived SH2 domain, miniprotein. C-type lectin-like domain scaffold,engineered antibody mimic, and any genetically manipulated counterpartsof any of the foregoing which retain binding functionality.

In certain embodiments, each of the two or more binding regions of themultivalent cell-targeting molecule comprises a polypeptide comprisingan immunoglobulin-type binding region and/or a polypeptide selected fromthe group consisting of: an autonomous V_(H) domain, single-domainantibody fragment (sdAb), nanobody, heavy chain-antibody domain derivedfrom a camelid (V_(H)H or V_(H) domain fragment), heavy-chain antibodydomain derived from a cartilaginous fish (V_(H)H or V_(H) domainfragment), immunoglobulin new antigen receptor (IgNAR), V_(NAR)fragment, single-chain variable fragment (scFv), antibody variablefragment (Fv), complementary determining region 3 fragment (CDR3),constrained FR3-CDR3-FR4 polypeptide (FR3-CDR3-FR4). Fd fragment, smallmodular immunopharmaceutical (SMIP) domain, antigen-binding fragment(Fab), Armadillo repeat polypeptide (ArmRP), fibronectin-derived 10^(th)fibronectin type III domain (10Fn3), tenascin type III domain (TNfn3),ankyrin repeat motif domain, low-density-lipoprotein-receptor-derivedA-domain (LDLR-A), lipocalin (anticalin). Kunitz domain,Protein-A-derived Z domain, gamma-B crystallin-derived domain,ubiquitin-derived domain, Sac7d-derived polypeptide (affitin),Fyn-derived SH2 domain, miniprotein, C-type lectin-like domain scaffold,engineered antibody mimic, and any genetically manipulated counterpartsof any of the foregoing which retain binding functionality.

In certain embodiments, the multivalent cell-targeting molecule of thepresent invention comprises two or more proteinaceous components (e.g.protein subunits), wherein each proteinaceous component comprises (a) atleast one of the two or more binding regions, and, optionally, (b) oneor more of the at least one, de-immunized, Shiga toxin effectorpolypeptide. In certain further embodiments, each proteinaceouscomponent comprises (1) only one of the two or more binding regions and(2) only one, de-immunized. Shiga toxin effector polypeptide. In certainfurther embodiments, the multivalent cell-targeting molecule of thepresent invention comprises exactly two proteinaceous components.

In certain embodiments, the multivalent cell-targeting molecule of thepresent invention comprises two or more components, wherein at least onecomponent is associated with the multivalent cell-targeting moleculethrough one or more non-covalent interactions. In certain furtherembodiments, at least one of the components is proteinaceous. In certainfurther embodiments, the multivalent cell-targeting molecule of thepresent invention comprises two or more proteinaceous componentsassociated with each other, either directly or indirectly, through oneor more non-covalent interactions. In certain further embodiments, eachproteinaceous component comprises (1) at least one of the two or morebinding regions and (2) at least one of the at least one, de-immunized,Shiga toxin effector polypeptide.

In certain embodiments, the multivalent cell-targeting molecule of thepresent invention comprises two or more Shiga toxin effectorpolypeptides, whether de-immunized or not de-immunized. In certainembodiments, the multivalent cell-targeting molecule of the presentinvention comprises two or more proteinaceous components (e.g. proteinsubunits), wherein each proteinaceous component comprises (a) at leastone of the two or more binding regions, and, optionally, (b) one or moreShiga toxin effector polypeptides. In certain further embodiments, eachproteinaceous component comprises (1) only one of the two or morebinding regions and (2) only one Shiga toxin effector polypeptide. Incertain further embodiments, the multivalent cell-targeting molecule ofthe present invention comprises two or more components (e.g. aproteinaceous component), wherein at least one component is associatedwith the multivalent cell-targeting molecule through one or morenon-covalent interactions. In certain further embodiments, eachproteinaceous component comprises (1) at least one of the two or morebinding regions and (2) at least one Shiga toxin effector polypeptide.

In certain embodiments, a Shiga toxin effector polypeptide component ofthe multivalent cell-targeting molecule of the present inventioncomprises a Shiga toxin A1 fragment region and/or Shiga toxin A1fragment derived region having a carboxy-terminus. In certain furtherembodiments, all of the Shiga toxin effector polypeptide components ofthe multivalent cell-targeting molecule each comprises a Shiga toxin A1fragment region and/or Shiga toxin A1 fragment derived region having acarboxy-terminus.

For certain embodiments of Embodiment Set #11, the multivalentcell-targeting molecule of the present invention is capable ofexhibiting (i) a catalytic activity level comparable to a wild-typeShiga toxin A1 fragment or wild-type Shiga toxin effector polypeptide,(ii) a ribosome inhibition activity with a half-maximal inhibitoryconcentration (IC₅₀) value of 10,000 picomolar or less, and/or (iii) asignificant level of Shiga toxin catalytic activity.

For certain embodiments of Embodiment Set #11, the multivalentcell-targeting molecule of the present invention and/or its at leastone, de-immunized, Shiga toxin effector polypeptide is capable ofexhibiting subcellular routing efficiency comparable to a referencecell-targeting molecule comprising a wild-type Shiga toxin A1 fragmentor wild-type Shiga toxin effector polypeptide and/or capable ofexhibiting a significant level of intracellular routing activity to theendoplasmic reticulum and/or cytosol from an endosomal starting locationof a cell.

For certain embodiments of Embodiment Set #11, whereby administration ofthe multivalent cell-targeting molecule of the present invention to acell physically coupled with extracellular target biomolecule of themultivalent cell-targeting molecule's two or more binding regions, themultivalent cell-targeting molecule is capable of causing death of thecell. For certain further embodiments, administration of the multivalentcell-targeting molecule of the invention to two different populations ofcell types which differ with respect to the presence or level ofphysically coupled extracellular target biomolecule, the multivalentcell-targeting molecule is capable of causing cell death to thecell-types physically coupled with an extracellular target biomoleculeof the multivalent cell-targeting molecule's two or more binding regionsat a CD₅₀ at least three times or less than the CD₅₀ to cell types whichare not physically coupled with an extracellular target biomolecule ofthe multivalent cell-targeting molecule's two or more binding regions.For certain embodiments, whereby administration of the multivalentcell-targeting molecule of the present invention to a first populationof cells whose members are physically coupled to extracellular targetbiomolecules of the multivalent cell-targeting molecule's two or morebinding regions, and a second population of cells whose members are notphysically coupled to any extracellular target biomolecule of the two ormore binding regions, the cytotoxic effect of the multivalentcell-targeting molecule to members of said first population of cellsrelative to members of said second population of cells is at least3-fold greater. For certain embodiments, whereby administration of themultivalent cell-targeting molecule of the present invention to a firstpopulation of cells whose members are physically coupled to asignificant amount of the extracellular target biomolecule of themultivalent cell-targeting molecule's two or more binding regions, and asecond population of cells whose members are not physically coupled to asignificant amount of any extracellular target biomolecule of the two ormore binding regions, the cytotoxic effect of the multivalentcell-targeting molecule to members of said first population of cellsrelative to members of said second population of cells is at least3-fold greater. For certain embodiments, whereby administration of themultivalent cell-targeting molecule of the present invention to a firstpopulation of target biomolecule positive cells, and a second populationof cells whose members do not express a significant amount of a targetbiomolecule of the multivalent cell-targeting molecule's two or morebinding regions at a cellular surface, the cytotoxic effect of themultivalent cell-targeting molecule to members of the first populationof cells relative to members of the second population of cells is atleast 3-fold greater.

For certain embodiments of Embodiment Set #11, the multivalentcell-targeting molecule of the present invention is capable whenintroduced to cells of exhibiting a cytotoxicity with a half-maximalinhibitory concentration (CD₅₀) value of 300 nanomolar (nM) or lessand/or capable of exhibiting a significant level of Shiga toxincytotoxicity.

For certain embodiments of Embodiment Set #11, the multivalentcell-targeting molecule of the present invention is capable ofdelivering an embedded or inserted, heterologous, CD8+ T-cell epitope toa MHC class I presentation pathway of a cell for cell-surfacepresentation of the epitope bound by a MHC class I molecule.

In certain embodiments of Embodiment Set #11, the multivalentcell-targeting molecule comprises a molecular moiety associated with thecarboxy-terminus of the at least one, de-immunized. Shiga toxin effectorpolypeptide. In certain embodiments, the molecular moiety comprises orconsists of the one or more the two or more binding regions. In certainembodiments, the molecular moiety comprises at least one amino acid andthe at least one, de-immunized, Shiga toxin effector polypeptide islinked, either directly or indirectly, to at least one amino acidresidue of the molecular moiety. In certain further embodiments, themolecular moiety and the at least one, de-immunized, Shiga toxineffector polypeptide are fused, either directly or indirectly, forming acontinuous polypeptide. In certain further embodiments, the molecularmoiety(ies) is each fused to the at least one, de-immunized, Shiga toxineffector polypeptide, either directly or indirectly, to form acontinuous polypeptide.

In certain embodiments of Embodiment Set #11, the multivalentcell-targeting molecule further comprises a cytotoxic molecular moietyassociated with the carboxy-terminus of the at least one, de-immunized,Shiga toxin effector polypeptide. In certain embodiments, the cytotoxicmolecular moiety is a cytotoxic agent, such as, e.g., a small moleculechemotherapeutic agent, anti-neoplastic agent, cytotoxic antibiotic,cytotoxic anti-infective, alkylating agent, antimetabolite,topoisomerase inhibitor, and/or tubulin inhibitor known to the skilledworker and/or described herein. In certain further embodiments, thecytotoxic molecular moiety is cytotoxic at concentrations of less than10,000, 5,000, 1,000, 500, or 200 picomolar (pM).

For certain embodiments of Embodiment Set #11, the two or more bindingregions are each capable of binding to an extracellular targetbiomolecule selected from the group consisting of: CD20, CD22, CD40,CD74, CD79, CD25, CD30, HER2/neu/ErbB2, EGFR, EpCAM, EphB2,prostate-specific membrane antigen, Cripto, CDCP1, endoglin, fibroblastactivated protein, Lewis-Y, CD19, CD21, CS1/SLAMF7, CD33, CD52, CD133,CEA, gpA33, mucin. TAG-72, tyrosine-protein kinase transmembranereceptor (ROR1 or NTRKR1), carbonic anhydrase IX, folate bindingprotein, ganglioside GD2, ganglioside GD3, ganglioside GM2, gangliosideLewis-Y2, VEGFR, Alpha V beta3, AlphaSbeta1, ErbB1/EGFR, Erb3, c-MET.IGF1R, EphA3, TRAIL-R1, TRAIL-R2, RANK, FAP, tenascin, CD64, mesothelin,BRCA1, MART-1/MelanA, gp100, tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3,GAGE-1/2, BAGE, RAGE, NY-ESO-1, CDK-4, beta-catenin, MUM-1, caspase-8,KIAA0205, HPVE6, SART-1, PRAME, carcinoembryonic antigen, prostatespecific antigen, prostate stem cell antigen, human aspartyl(asparaginyl) beta-hydroxylase, EphA2, HER3/ErbB-3, MUC1, MART-1/MelanA,gp100, tyrosinase associated antigen, HPV-E7, Epstein-Barr virusantigen, Bcr-Abl, alpha-fetoprotein antigen, 17-A1, bladder tumorantigen, SAIL, CD38, CD15, CD23, CD45 (protein tyrosine phosphatasereceptor type C), CD53, CD88, CD129, CD183, CD191, CD193, CD244, CD294,CD305, C3AR, FceRIa, galectin-9, IL-1R (interleukin-1 receptor), mrp-14,NKG2D ligand, programmed death-ligand 1 (PD-L1), Siglec-8, Siglec-10,CD49d, CD13, CD44, CD54, CD63, CD69, CD123, TLR4, FceRIa, IgE, CD107a,CD203c, CD14, CD68, CD80, CD86, CD105, CD115, F4/80, ILT-3, galectin-3,CD11a-c, GITRL, MHC class I molecule, MHC class II molecule (optionallycomplexed with a peptide), CD284 (TLR4), CD107-Mac3, CD195 (CCR5),HLA-DR, CD16/32, CD282 (TLR2), CD11c, and any immunogenic fragment ofany of the foregoing.

In certain embodiments of Embodiment Set #11, one or more of the two ormore binding regions is linked, either directly or indirectly, to atleast one of the at least one, de-immunized, Shiga toxin effectorpolypeptide by at least one covalent bond which is not a disulfide bond.In certain further embodiments, one or more of the multivalentcell-targeting molecule's two or more binding regions is fused, eitherdirectly or indirectly, to the carboxy-terminus of the at least one,de-immunized, Shiga toxin effector polypeptide to form a single,continuous polypeptide. In certain further embodiments, at least one ofthe two or more binding regions comprises an immunoglobulin-type bindingregion. In certain further embodiments, all of the multivalentcell-targeting molecule's two or more binding regions each comprises animmunoglobulin-type binding region.

In certain embodiments of Embodiment Set #11, the disruptedfurin-cleavage motif comprises one or more mutations in the minimal,furin-cleavage site relative to a wild-type Shiga toxin A Subunit. Incertain embodiments, the disrupted furin-cleavage motif is not anamino-terminal truncation of sequences that overlap with part or all ofat least one amino acid residue of the minimal furin-cleavage site. Incertain embodiments, the mutation in the minimal, furin-cleavage site isan amino acid deletion, insertion, and/or substitution of at least oneamino acid residue in the R/Y-x-x-R furin cleavage motif. In certainfurther embodiments, the disrupted furin-cleavage motif comprises atleast one mutation relative to a wild-type Shiga toxin A Subunit, themutation altering at least one amino acid residue in the region nativelypositioned at 248-251 of the A Subunit of Shiga toxin (SEQ ID NOs: 1-2and 4-6), or at 247-250 of the A Subunit of Shiga-like toxin 2 (SEQ IDNOs: 3 and 7-18), or the equivalent amino acid sequence position in anyShiga toxin A Subunit.

In certain further embodiments, the mutation is an amino acid residuesubstitution of an arginine residue with a non-positively charged, aminoacid residue.

In certain embodiments of Embodiment Set #11, at least one of the two ormore binding regions comprises the peptide or polypeptide shown in anyone of SEQ ID NOs: 39-245.

In certain embodiments of Embodiment Set #11, the multivalentcell-targeting molecule of the present invention comprises thepolypeptide shown in any one of SEQ ID NOs: 252-255 and 288-748. Incertain further embodiments, the multivalent cell-targeting molecule ofthe present invention comprises or consists essentially of two proteins,each protein selected from any one of the polypeptides shown in SEQ IDNOs: 252-255 and 288-748, and optionally, each protein further comprisesan amino-terminal methionine residue.

In certain embodiments of Embodiment Set #11, at least one of the two ormore binding regions sterically covers the carboxy-terminus of the A1fragment region or Shiga toxin A1 fragment derived region of at leastone of the at least one, de-immunized, Shiga toxin effectorpolypeptide(s). In certain further embodiments, the at least one of thetwo or more binding regions sterically cover the carboxy-terminals ofthe A1 fragment region or A1 fragment derived region of all the Shigatoxin effector polypeptide component(s) present in the multivalentcell-targeting molecule. In certain further embodiments, each of thecarboxy-terminals of the A1 fragment region or A1 fragment derivedregion of each of the Shiga toxin effector polypeptide componentspresent in the multivalent cell-targeting molecule is sterically coveredby at least one of the two or more binding regions.

In certain embodiments of Embodiment Set #11, the molecular moietysterically covers the carboxy-terminus of the A1 fragment region and/orShiga toxin A1 fragment derived region of at least one of the at leastone, de-immunized, Shiga toxin effector polypeptide(s). In certainfurther embodiments, the molecular moiety comprises at least one of thetwo or more binding regions.

In certain embodiments of Embodiment Set #11, the molecular moietysterically covers the carboxy-terminus of the A1 fragment region and/orShiga toxin A1 fragment derived region of at least one of the at leastone, de-immunized, Shiga toxin effector polypeptide(s). In certainfurther embodiments, the molecular moiety(ies) sterically cover thecarboxy-terminals of the A1 fragment region or A1 fragment derivedregion of all the Shiga toxin effector polypeptide component(s) presentin the multivalent cell-targeting molecule. In certain furtherembodiments, each of the carboxy-terminals of the A1 fragment region orA1 fragment derived region of each of the Shiga toxin effectorpolypeptide components present in the multivalent cell-targetingmolecule is sterically covered by at least one of the molecularmoiety(ies). In certain further embodiments, each of the molecularmoieties present in the multivalent cell-targeting molecule comprises atleast one of the two or more binding regions.

In certain embodiments of Embodiment Set #11, the multivalentcell-targeting molecule of the present invention comprises at least oneof the two or more binding regions and/or molecular moiety locatedcarboxy-terminal to the carboxy-terminus of the Shiga toxin A1 fragmentregion and/or Shiga toxin A1 fragment derived region of the at leastone, de-immunized, Shiga toxin effector polypeptide.

In certain further embodiments, the mass of the binding region and/ormolecular moiety is at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20kDa, 25 kDa, 28 kDa, kDa, 41 kDa, 50 kDa, 100 kDa, or greater. Incertain embodiments, the multivalent cell-targeting molecule of thepresent invention comprises the two or more binding regions and/or themolecular moiety located within the multivalent cell-targeting moleculeat a position carboxy-terminal to the carboxy-terminus of the Shigatoxin A1 fragment region and/or Shiga toxin A1 fragment derived regionof the at least one, de-immunized, Shiga toxin effector polypeptide.

In certain embodiments of Embodiment Set #11, the multivalentcell-targeting molecule comprises the binding region with a mass of atleast 4.5 kDa, 6, kDa, 9 kDa, 12 kDa 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30kDa, 41 kDa, 50 kDa, 100 kDa, or greater, as long as the multivalentcell-targeting molecule retains the appropriate level of the Shiga toxinbiological activity noted herein (e.g., cytotoxicity, intracellularrouting, and/or cellular internalization kinetic parameter(s)). Incertain embodiments of Embodiment Set #11, the two or more bindingregions have a combined mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa,kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, orgreater, as long as the multivalent cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein(e.g., cytotoxicity, intracellular routing, and/or cellularinternalization kinetic parameter(s)). In certain further embodiments,each of the two or more binding regions of the multivalentcell-targeting molecule comprises a binding region with a mass of atleast 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, kDa, 20 kDa, 25 kDa, 28 kDa, 30kDa, 41 kDa, 50 kDa, 100 kDa, or greater, as long as the multivalentcell-targeting molecule retains the appropriate level of the Shiga toxinbiological activity noted herein (e.g., cytotoxicity, intracellularrouting, and/or cellular internalization kinetics parameter(s)).

In certain embodiments of Embodiment Set #11, at least one of the two ormore binding regions is comprised within a relatively large, molecularmoiety such as, e.g., the molecular moiety having a mass of at least 4.5kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41kDa, 50 kDa, 100 kDa, or greater, as long as the multivalentcell-targeting molecule retains the appropriate level of the Shiga toxinbiological activity noted herein.

In certain embodiments of Embodiment Set #11, at least one,de-immunized, Shiga toxin effector polypeptide is more proximal to anamino-terminus of a polypeptide component of the multivalentcell-targeting molecule relative to any of the two or more bindingregions. In certain further embodiments, none of the two or more bindingregions comprised within the same polypeptide chain of a component ofthe multivalent cell-targeting molecule comprising at least one,de-immunized, Shiga toxin effector polypeptide, are located proximal toan amino-terminus of that polypeptide chain relative to at least one,de-immunized, Shiga toxin effector polypeptide comprised within thatpolypeptide chain. In certain embodiments, the amino-terminus of atleast one, de-immunized, Shiga toxin effector polypeptide is at and/orproximal to an amino-terminus of a polypeptide component of themultivalent cell-targeting molecule. In certain further embodiments, theamino-terminus of all the Shiga toxin effector polypeptides present inthe multivalent cell-targeting molecule is at and/or proximal to anamino-terminus of a polypeptide component of the multivalentcell-targeting molecule. In certain further embodiments, the two or morebinding regions and the least one, de-immunized, Shiga toxin effectorpolypeptide are physically arranged or oriented within the multivalentcell-targeting molecule such that none of the two or more bindingregions are located proximal to the amino-terminus of at least one,de-immunized. Shiga toxin effector polypeptide relative to that Shigatoxin effector polypeptide component's carboxy-terminus. In certainfurther embodiments, none of the two or more binding regions are locatedproximal to any amino-terminus of the multivalent cell-targetingmolecule relative to at least one Shiga toxin effector polypeptidecomponent. In certain embodiments, the two or more binding regions arelinked within the multivalent cell-targeting molecule more proximal tothe carboxy-terminus of the at least one, de-immunized. Shiga toxineffector polypeptide than to the amino-terminus of that de-immunized,Shiga toxin effector polypeptide. In certain embodiments, all thede-immunized. Shiga toxin effector polypeptide components are moreproximal to an amino-terminus of a polypeptide component of themultivalent cell-targeting molecule relative to any of the two or morebinding regions comprised within the same polypeptide chain of thatpolypeptide component. In certain further embodiments, the two or morebinding regions and the least one, de-immunized, Shiga toxin effectorpolypeptide are physically arranged or oriented within the multivalentcell-targeting molecule such that none of the two or more bindingregions are located proximal to the amino-terminus of any de-immunized,Shiga toxin effector polypeptide component relative to that Shiga toxineffector polypeptide component's carboxy-terminus. In certainembodiments, all the Shiga toxin effector polypeptide components aremore proximal to an amino-terminus of a polypeptide component of themultivalent cell-targeting molecule relative to any of the two or morebinding regions comprised within the same polypeptide chain of thatpolypeptide component. In certain further embodiments, the two or morebinding regions are physically arranged or oriented within themultivalent cell-targeting molecule such that none of the two or morebinding regions are located proximal to the amino-terminus of any Shigatoxin effector polypeptide component relative to that Shiga toxineffector polypeptide component's carboxy-terminus. For certain furtherembodiments, the multivalent cell-targeting molecule of the presentinvention is capable when introduced to cells of exhibiting cytotoxicitythat is greater than that of a reference molecule, such as, e.g., afifth cell-targeting molecule comprising a polypeptide component havingan amino-terminus and comprising the same two or more binding regionsand the same de-immunized, Shiga toxin effector polypeptide(s) which isnot positioned at or proximal to a physical amino-terminus of apolypeptide component of the fifth cell-targeting molecule or a sixthcell-targeting molecule comprising the same two or more binding regionsand the same Shiga toxin effector polypeptide component(s) as themultivalent cell-targeting molecule wherein at least one of the two ormore binding regions is more proximal to an amino-terminus of apolypeptide component of the multivalent cell-targeting moleculerelative to all the Shiga toxin effector polypeptide components. Forcertain further embodiments, the cell-targeting molecule of the presentinvention exhibits cytotoxicity with better optimized, cytotoxicpotency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greatercytotoxicity as compared to the cytotoxicity of the fourthcell-targeting molecule. For certain further embodiments, thecytotoxicity of the cell-targeting molecule of the present invention toa population of target positive cells is 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold or greater than the cytotoxicity of thefifth and/or sixth cell-targeting molecule to a second population oftarget positive cells as assayed by CD₅₀ values. In certain furtherembodiments, the multivalent cell-targeting molecule of the presentinvention is not cytotoxic and is capable when introduced to cells ofexhibiting a greater subcellular routing efficiency from anextracellular space to a subcellular compartment of an endoplasmicreticulum and/or cytosol as compared to the subcellular routingefficiency of the fifth and/or sixth cell-targeting molecule. In certainfurther embodiments, the fifth and/or sixth cell-targeting molecule doesnot comprise any carboxy-terminal, endoplasmic reticulumretention/retrieval signal motif of the KDEL family.

In certain embodiments of Embodiment Set #11, the amino-terminus of theat least one, de-immunized, Shiga toxin effector polypeptide is atand/or proximal to an amino-terminus of a polypeptide component of themultivalent cell-targeting molecule. In certain further embodiments, theamino-terminus of all the Shiga toxin effector polypeptides present inthe multivalent cell-targeting molecule is at and/or proximal to anamino-terminus of a polypeptide component of the multivalentcell-targeting molecule. In certain further embodiments, the at leastone, de-immunized, Shiga toxin effector polypeptide is more proximal toan amino-terminus of a polypeptide component of the multivalentcell-targeting molecule relative to any of the two or more bindingregions. In certain further embodiments, none of the two or more bindingregions comprised within the same polypeptide chain of a polypeptidecomponent of the multivalent cell-targeting molecule comprising at leastone, de-immunized, Shiga toxin effector polypeptide, are locatedproximal to an amino-terminus of that polypeptide chain relative to atleast one, de-immunized, Shiga toxin effector polypeptide comprisedwithin that polypeptide chain. In certain further embodiments, the twoor more binding regions and the least one, de-immunized. Shiga toxineffector polypeptide are physically arranged or oriented within themultivalent cell-targeting molecule such that none of the two or morebinding regions are located proximal to the amino-terminus of at leastone, de-immunized, Shiga toxin effector polypeptide relative to thatShiga toxin effector polypeptide component's carboxy-terminus. Incertain further embodiments, none of the two or more binding regions arelocated proximal to any amino-terminus of the multivalent cell-targetingmolecule relative to at least one Shiga toxin effector polypeptidecomponent. In certain embodiments, the two or more binding regions arelinked within the multivalent cell-targeting molecule more proximal tothe carboxy-terminus of the at least one, de-immunized, Shiga toxineffector polypeptide than to the amino-terminus of that de-immunized,Shiga toxin effector polypeptide. In certain further embodiments, noneof the two or more binding regions are located proximal to anyamino-terminus of the multivalent cell-targeting molecule relative to atleast one Shiga toxin effector polypeptide component. In certain furtherembodiments, the two or more binding regions are linked within themultivalent cell-targeting molecule more proximal to thecarboxy-terminus of the at least one Shiga toxin effector polypeptidethan to the amino-terminus of that Shiga toxin effector polypeptide. Incertain embodiments, all the de-immunized, Shiga toxin effectorpolypeptide components are more proximal to an amino-terminus of apolypeptide component of the multivalent cell-targeting moleculerelative to any of the two or more binding regions comprised within thesame polypeptide chain of that polypeptide component. In certain furtherembodiments, the two or more binding regions and the least one,de-immunized, Shiga toxin effector polypeptide are physically arrangedor oriented within the multivalent cell-targeting molecule such thatnone of the two or more binding regions are located proximal to theamino-terminus of any de-immunized, Shiga toxin effector polypeptidecomponent relative to that Shiga toxin effector polypeptide component'scarboxy-terminus. In certain embodiments, all the Shiga toxin effectorpolypeptide components are more proximal to an amino-terminus of apolypeptide component of the multivalent cell-targeting moleculerelative to any of the two or more binding regions comprised within thesame polypeptide chain of that polypeptide component. In certain furtherembodiments, the two or more binding regions are physically arranged ororiented within the multivalent cell-targeting molecule such that noneof the two or more binding regions are located proximal to theamino-terminus of any Shiga toxin effector polypeptide componentrelative to that Shiga toxin effector polypeptide component'scarboxy-terminus. For certain embodiments, the cell-targeting moleculeof the present invention exhibits cytotoxicity with better optimized,cytotoxic potency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, orgreater cytotoxicity as compared to the cytotoxicity of a referencemolecule, such as, e.g., a seventh multivalent cell-targeting moleculehaving an amino-terminus and comprising the same two or more bindingregions and the same Shiga toxin effector polypeptide component(s) asthe multivalent cell-targeting molecule wherein none of the Shiga toxineffector polypeptide components is at and/or proximal to anamino-terminus of a polypeptide component of the multivalentcell-targeting molecule. For certain further embodiments, thecytotoxicity of the cell-targeting molecule of the present invention toa population of target positive cells is 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold or greater than the cytotoxicity of theseventh cell-targeting molecule to a second population of targetpositive cells as assayed by CD₅₀ values. In certain furtherembodiments, the multivalent cell-targeting molecule of the presentinvention is not cytotoxic and is capable when introduced to cells ofexhibiting a greater subcellular routing efficiency from anextracellular space to a subcellular compartment of an endoplasmicreticulum and/or cytosol as compared to the subcellular routingefficiency of the seventh cell-targeting molecule. In certain furtherembodiments, the seventh cell-targeting molecule does not comprise anycarboxy-terminal, endoplasmic reticulum retention/retrieval signal motifof the KDEL family.

In certain embodiments of Embodiment Set #11, the two or more bindingregions are not located proximal to an amino-terminus of a polypeptidecomponent of the multivalent cell-targeting molecule relative to atleast one, de-immunized, Shiga toxin effector polypeptide. In certainfurther embodiments, the two or more binding regions and Shiga toxineffector polypeptide are physically arranged or oriented within themultivalent cell-targeting molecule such that the two or more bindingregion are not located proximal to the amino-terminus of at least one,de-immunized, Shiga toxin effector polypeptide. In certain furtherembodiments, none of the two or more binding regions comprised withinthe same polypeptide chain of a component of the multivalentcell-targeting molecule comprising at least one, de-immunized, Shigatoxin effector polypeptide, are located proximal to an amino-terminus ofthat polypeptide chain relative to at least one, de-immunized, Shigatoxin effector polypeptide comprised within that polypeptide chain. Incertain further embodiments, the amino-terminus of at least one,de-immunized, Shiga toxin effector polypeptide is at and/or proximal toan amino-terminus of a polypeptide component of the multivalentcell-targeting molecule. In certain further embodiments, theamino-terminus of all the Shiga toxin effector polypeptides present inthe multivalent cell-targeting molecule is at and/or proximal to anamino-terminus of a polypeptide component of the multivalentcell-targeting molecule. In certain further embodiments, the two or morebinding regions and the least one, de-immunized, Shiga toxin effectorpolypeptide are physically arranged or oriented within the multivalentcell-targeting molecule such that none of the two or more bindingregions are located proximal to the amino-terminus of at least one,de-immunized, Shiga toxin effector polypeptide relative to that Shigatoxin effector polypeptide component's carboxy-terminus. In certainfurther embodiments, none of the two or more binding regions are locatedproximal to any amino-terminus of the multivalent cell-targetingmolecule relative to at least one Shiga toxin effector polypeptidecomponent. In certain embodiments, the two or more binding regions arelinked within the multivalent cell-targeting molecule more proximal tothe carboxy-terminus of the at least one, de-immunized, Shiga toxineffector polypeptide than to the amino-terminus of that de-immunized.Shiga toxin effector polypeptide. In certain embodiments, all thede-immunized, Shiga toxin effector polypeptide components are moreproximal to an amino-terminus of a polypeptide component of themultivalent cell-targeting molecule relative to any of the two or morebinding regions comprised within the same polypeptide chain of thatpolypeptide component. In certain further embodiments, the two or morebinding regions and the least one, de-immunized, Shiga toxin effectorpolypeptide are physically arranged or oriented within the multivalentcell-targeting molecule such that none of the two or more bindingregions are located proximal to the amino-terminus of any de-immunized,Shiga toxin effector polypeptide component relative to that Shiga toxineffector polypeptide component's carboxy-terminus. In certainembodiments, all the Shiga toxin effector polypeptide components aremore proximal to an amino-terminus of a polypeptide component of themultivalent cell-targeting molecule relative to any of the two or morebinding regions comprised within the same polypeptide chain of thatpolypeptide component. In certain further embodiments, the two or morebinding regions are physically arranged or oriented within themultivalent cell-targeting molecule such that none of the two or morebinding regions are located proximal to the amino-terminus of any Shigatoxin effector polypeptide component relative to that Shiga toxineffector polypeptide component's carboxy-terminus. For certainembodiments, the cell-targeting molecule of the present inventionexhibits cytotoxicity with better optimized, cytotoxic potency, such as,e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity ascompared to the cytotoxicity of a reference molecule, such as, e.g., aneighth multivalent cell-targeting molecule having polypeptide componenthaving an amino-terminus and comprising the same two or more bindingregions and the same Shiga toxin effector polypeptide component(s) asthe multivalent cell-targeting molecule wherein at least one of the twoor more binding regions is located proximal to an amino-terminus of apolypeptide component of the multivalent cell-targeting moleculerelative to each of all the Shiga toxin effector polypeptidecomponent(s). In certain further embodiments, the multivalentcell-targeting molecule of the present invention is not cytotoxic and iscapable when introduced to cells of exhibiting a greater subcellularrouting efficiency from an extracellular space to a subcellularcompartment of an endoplasmic reticulum and/or cytosol as compared tothe subcellular routing efficiency of the eighth cell-targetingmolecule. In certain further embodiments, the eighth cell-targetingmolecule does not comprise any carboxy-terminal, endoplasmic reticulumretention/retrieval signal motif of the KDEL family.

In certain embodiments of Embodiment Set #11, the multivalentcell-targeting molecule of the present invention, and/or a polypeptidecomponent thereof, comprises a carboxy-terminal, endoplasmic reticulumretention/retrieval signal motif of a member of the KDEL family. Incertain further embodiments, the carboxy-terminal endoplasmic reticulumretention/retrieval signal motif is selected from the group consistingof KDEL. HDEF, HDEL. RDEF. RDEL, WDEL, YDEL, HEEF, HEEL, KEEL, REEL,KAEL, KCEL, KFEL, KGEL, KHEL, KLEL, KNEL, KQEL, KREL, KSEL, KVEL, KWEL,KYEL, KEDL, KIEL, DKEL, FDEL, KDEF, KKEL, HADL, HAEL, HIEL, HNEL, HTEL,KTEL, HVEL, NDEL, QDEL, REDL, RNEL, RTDL. RTEL, SDEL, TDEL, SKEL, STEL,and EDEL. In certain further embodiments, the multivalent cell-targetingmolecule of the present invention is capable when introduced to cells ofexhibiting cytotoxicity that is greater than that of a referencemolecule, such as, e.g., a ninth cell-targeting molecule consisting ofthe cell-targeting molecule except for it does not comprise anycarboxy-terminal, endoplasmic reticulum retention/retrieval signal motifof the KDEL family. In certain further embodiments, the cell-targetingmolecule of the present invention is capable of exhibiting acytotoxicity with better optimized, cytotoxic potency, such as, e.g.,4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity as compared to areference molecule, such as, e.g., the ninth cell-targeting molecule. Incertain further embodiments, the cytotoxicity of the cell-targetingmolecule of the present invention to a population of target positivecells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-foldor greater than the cytotoxicity of the ninth cell-targeting molecule toa second population of target positive cells as assayed by CD₅₀ values.

For certain further embodiments, the multivalent cell-targeting moleculeof the present invention exhibits low cytotoxic potency (i.e. is notcapable when introduced to certain positive target cell types ofexhibiting a cytotoxicity greater than 1% cell death of a cellpopulation at a multivalent cell-targeting molecule concentration of1000 nM, 500 nM, 100 nM, 75 nM, or 50 nM) and is capable when introducedto cells of exhibiting a greater subcellular routing efficiency from anextracellular space to a subcellular compartment of an endoplasmicreticulum and/or cytosol as compared to the subcellular routingefficiency of the second, third, fourth, fifth, sixth, seventh, eighth,and/or ninth cell-targeting molecule.

Embodiment Set #12—Multivalent Cell-Targeting Molecules Comprising aCarboxy-Terminal Endoplasmic Reticulum Retention/Retrieval Signal Motifand a Shiga Toxin Effector Polypeptide Comprising an Embedded orInserted, Heterologous, T-Cell Epitope

The present invention provides multivalent cell-targeting molecules,each comprising (i) two or more binding regions, each capable ofspecifically binding an extracellular part of the same targetbiomolecule; (ii) a Shiga toxin effector polypeptide comprising aninserted or embedded, heterologous epitope; and (iii) acarboxy-terminal, endoplasmic reticulum retention/retrieval signalmotif. In certain embodiments, the cell-targeting molecule of thepresent invention comprises (i) two or more binding regions, eachcapable of specifically binding an extracellular target biomolecule;(ii) a Shiga toxin effector polypeptide comprising an embedded orinserted, heterologous epitope; and (iii) a carboxy-terminal,endoplasmic reticulum retention/retrieval signal motif of a member ofthe KDEL family. For certain further embodiments, the Shiga toxineffector polypeptide is capable of exhibiting at least one Shiga toxineffector function, such as, e.g., directing intracellular routing to theendoplasmic reticulum and/or cytosol of a cell in which the polypeptideis present, inhibiting a ribosome function, enzymatically inactivating aribosome, causing cytostasis, and/or causing cytotoxicity. In certainembodiments, the heterologous epitope is a CD8+ T-cell epitope, such as,e.g., with regard to a human immune system. For certain furtherembodiments, the heterologous, CD8+ T-cell epitope is capable of beingpresented by a MHC class I molecule of a cell. For certain embodimentsof Embodiment Set #12, the cell-targeting molecule of the presentinvention is capable of one or more the following: entering a cell,inhibiting a ribosome function, causing cytostasis, causing cell death,and/or delivering the embedded or inserted, heterologous T-cell epitopeto a MHC class I molecule for presentation on a cellular surface.

In certain embodiments of Embodiment Set #12, the carboxy-terminalendoplasmic reticulum retention/retrieval signal motif is selected fromthe group consisting of: KDEL, HDEF, HDEL, RDEF, RDEL, WDEL, YDEL, HEEF,HEEL, KEEL, REEL, KAEL, KCEL, KFEL, KGEL, KHEL, KLEL, KNEL, KQEL, KREL,KSEL, KVEL, KWEL, KYEL, KEDL, KIEL, DKEL, FDEL, KDEF, KKEL, HADL, HAEL,HIEL, HNEL, HTEL, KTEL, HVEL, NDEL, QDEL, REDL, RNEL, RTDL, RTEL, SDEL,TDEL, SKEL, STEL, and EDEL.

In certain embodiments of Embodiment Set #12, the inserted or embedded,heterologous, T-cell epitope disrupts the endogenous, B-cell and/orT-cell epitope region selected from the group of natively positionedShiga toxin A Subunit regions consisting of: (i) 1-15 of any one of SEQID NOs: 1-2 and 4-6; 3-14 of any one of SEQ ID NOs: 3 and 7-18; 26-37 ofany one of SEQ ID NOs: 3 and 7-18; 27-37 of any one of SEQ ID NOs: 1-2and 4-6; 39-48 of any one of SEQ ID NOs: 1-2 and 4-6; 42-48 of any oneof SEQ ID NOs: 3 and 7-18; and 53-66 of any one of SEQ ID NOs: 1-18, orthe equivalent region in a Shiga toxin A Subunit or derivative thereof;(ii) 94-115 of any one of SEQ ID NOs: 1-18; 141-153 of any one of SEQ IDNOs: 1-2 and 4-6; 140-156 of any one of SEQ ID NOs: 3 and 7-18; 179-190of any one of SEQ ID NOs: 1-2 and 4-6; 179-191 of any one of SEQ ID NOs:3 and 7-18; 204 of SEQ ID NO:3; 205 of any one of SEQ ID NOs: 1-2 and4-6; and 210-218 of any one of SEQ ID NOs: 3 and 7-18, or the equivalentregion in a Shiga toxin A Subunit or derivative thereof: and (iii)240-260 of any one of SEQ ID NOs: 3 and 7-18; 243-257 of any one of SEQID NOs: 1-2 and 4-6; 254-268 of any one of SEQ ID NOs: 1-2 and 4-6;262-278 of any one of SEQ ID NOs: 3 and 7-18: 281-297 of any one of SEQID NOs: 3 and 7-18; and 285-293 of any one of SEQ ID NOs: 1-2 and 4-6,or the equivalent region in a Shiga toxin A Subunit or derivativethereof.

For certain further embodiments of Embodiment Set #12, the heterologousepitope is a CD8+ T-cell epitope capable of being presented by a MHCclass I molecule of a cell. In certain further embodiments, theheterologous epitope in is embedded and replaces an equivalent number ofamino acid residues in a wild-type Shiga toxin polypeptide region suchthat the Shiga toxin effector polypeptide has the same total number ofamino acid residues as does the wild-type Shiga toxin polypeptide regionfrom which it is derived. For certain further embodiments of any of theabove, the Shiga toxin effector polypeptide is capable of exhibiting atleast one Shiga toxin effector function selected from directingintracellular routing to a cytosol of a cell in which the polypeptide ispresent, inhibiting a ribosome function, enzymatically inactivating aribosome, and cytotoxicity.

In certain embodiments of Embodiment Set #12, the cell-targetingmolecule of the present invention is capable when introduced to cells ofexhibiting cytotoxicity that is greater than that of a fifthcell-targeting molecule consisting of the cell-targeting molecule exceptfor it does not comprise any carboxy-terminal, endoplasmic reticulumretention/retrieval signal motif of the KDEL family. In certain furtherembodiments, the cell-targeting molecule of the present invention iscapable of exhibiting a cytotoxicity with better optimized, cytotoxicpotency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greatercytotoxicity as compared to the fifth cell-targeting molecule. Incertain further embodiments, the cytotoxicity of the cell-targetingmolecule of the present invention to a population of target positivecells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-foldor greater than the cytotoxicity of the fifth cell-targeting molecule toa second population of target positive cells as assayed by CD₅₀ values.

For certain embodiments of Embodiment Set #12, the cell-targetingmolecule of the present invention is capable of delivering an embeddedor inserted, heterologous, CD8+ T-cell epitope to a MHC class Ipresentation pathway of a cell for cell-surface presentation of theepitope bound by a MHC class I molecule.

For certain embodiments of Embodiment Set #12, the cell-targetingmolecule is de-immunized due to the embedded or inserted, heterologousepitope. In certain further embodiments, the cell-targeting molecule iscapable of exhibiting less relative antigenicity and/or relativeimmunogenicity as compared to a reference molecule, such as, e.g., asixth cell-targeting molecule consisting of the cell-targeting moleculeexcept for it lacks one or more embedded or inserted epitopes present inone or more Shiga toxin effector polypeptide components of the celltargeting molecule.

For certain further embodiments of Embodiment Set #12, thecell-targeting molecule of the present invention is not cytotoxic and iscapable when introduced to cells of exhibiting a greater subcellularrouting efficiency from an extracellular space to a subcellularcompartment of an endoplasmic reticulum and/or cytosol as compared tothe subcellular routing efficiency of a reference molecule, such as,e.g., the fifth cell-targeting molecule.

Embodiment Set #13—Multivalent Cell-Targeting Molecules Comprising aShiga Toxin Effector Polypeptide Comprising (i) an Embedded or Inserted,Heterologous, T-Cell Epitope and (ii) a Disrupted. Furin-Cleavage Motifat the Carboxy-Terminus of an A1 Fragment Derived Region

The present invention provides multivalent cell-targeting molecules,each comprising (i) two or more binding regions, each capable ofspecifically binding an extracellular part of the same targetbiomolecule; (ii) a Shiga toxin effector polypeptide comprising aninserted or embedded, heterologous epitope; and (iii) a disruptedfurin-cleavage motif. In certain embodiments, the cell-targetingmolecule of the present invention comprises (i) a binding region capableof specifically binding an extracellular target biomolecule; (ii) aShiga toxin effector polypeptide comprising (a) an inserted or embedded,heterologous epitope; (b) a Shiga toxin A1 fragment derived regionhaving a carboxy terminus; and (c) a disrupted furin-cleavage motif atthe carboxy-terminus of the A1 fragment derived region. In certainembodiments, the cell-targeting molecule of the present inventioncomprises (i) a binding region capable of specifically binding anextracellular target biomolecule; (ii) a Shiga toxin effectorpolypeptide comprising (a) an inserted or embedded, heterologousepitope; (b) a Shiga toxin A1 fragment region having a carboxy terminus,and (c) a disrupted furin-cleavage motif at the carboxy-terminus of theA1 fragment region. For certain further embodiments, the Shiga toxineffector polypeptide is capable of exhibiting at least one Shiga toxineffector function, such as, e.g., directing intracellular routing to theendoplasmic reticulum and/or cytosol of a cell in which the polypeptideis present, inhibiting a ribosome function, enzymatically inactivating aribosome, causing cytostasis, and/or causing cytotoxicity. In certainfurther embodiments, the heterologous epitope is a CD8+ T-cell epitope,such as, e.g., with regard to a human immune system. For certain furtherembodiments, the heterologous, T-cell epitope is capable of beingpresented by a MHC class I molecule of a cell. In certain furtherembodiments, the cell-targeting molecule of the present invention iscapable of one or more the following: entering a cell, inhibiting aribosome function, causing cytostasis, causing cell death, and/ordelivering the embedded or inserted, heterologous, T-cell epitope to aMHC class I molecule for presentation on a cellular surface. For certainfurther embodiments, the cell-targeting molecule is capable whenintroduced to cells of exhibiting a cytotoxicity comparable or betterthan a reference molecule, such as, e.g., a second cell-targetingmolecule consisting of the cell-targeting molecule except for at leastone of its Shiga toxin effector polypeptide components comprise awild-type Shiga toxin furin-cleavage site at the carboxy terminus of itsA1 fragment region. For certain further embodiments, the cell-targetingmolecule is capable when introduced to cells of exhibiting acytotoxicity comparable or better than a second cell-targeting moleculeconsisting of the cell-targeting molecule except for all of its Shigatoxin effector polypeptide components comprise a wild-type Shiga toxinfurin-cleavage site at the carboxy terminus of its A1 fragment region.

In certain embodiments of Embodiment Set #13, the inserted or embedded,heterologous, epitope disrupts the endogenous, B-cell and/or T-cellepitope region selected from the group of natively positioned Shigatoxin A Subunit regions consisting of (i) 1-15 of any one of SEQ ID NOs:1-2 and 4-6; 3-14 of any one of SEQ ID NOs: 3 and 7-18; 26-37 of any oneof SEQ ID NOs: 3 and 7-18; 27-37 of any one of SEQ ID NOs: 1-2 and 4-6;39-48 of any one of SEQ ID NOs: 1-2 and 4-6; 42-48 of any one of SEQ IDNOs: 3 and 7-18; and 53-66 of any one of SEQ ID NOs: 1-18, or theequivalent region in a Shiga toxin A Subunit or derivative thereof; (ii)94-115 of any one of SEQ ID NOs: 1-18; 141-153 of any one of SEQ ID NOs:1-2 and 4-6; 140-156 of any one of SEQ ID NOs: 3 and 7-18; 179-190 ofany one of SEQ ID NOs: 1-2 and 4-6; 179-191 of any one of SEQ ID NOs: 3and 7-18; 204 of SEQ ID NO:3; 205 of any one of SEQ ID NOs: 1-2 and 4-6;and 210-218 of any one of SEQ ID NOs: 3 and 7-18, or the equivalentregion in a Shiga toxin A Subunit or derivative thereof; and (iii)240-260 of any one of SEQ ID NOs: 3 and 7-18; 243-257 of any one of SEQID NOs: 1-2 and 4-6; 254-268 of any one of SEQ ID NOs: 1-2 and 4-6;262-278 of any one of SEQ ID NOs: 3 and 7-18; 281-297 of any one of SEQID NOs: 3 and 7-18; and 285-293 of any one of SEQ ID NOs: 1-2 and 4-6,or the equivalent region in a Shiga toxin A Subunit or derivativethereof.

In certain embodiments of Embodiment Set #13, the disruptedfurin-cleavage motif comprises one or more mutations, relative to awild-type Shiga toxin A Subunit, the mutation altering at least oneamino acid residue in a region natively positioned at 248-251 of the ASubunit of Shiga toxin (SEQ ID NOs: 1-2 and 4-6), or at 247-250 of the ASubunit of Shiga-like toxin 2 (SEQ ID NOs: 3 and 7-18), or theequivalent region in a Shiga toxin A Subunit or derivative thereof. Incertain further embodiments, the disrupted furin-cleavage motifcomprises one or more mutations, relative to a wild-type Shiga toxin ASubunit, in a minimal furin cleavage site of the furin-cleavage motif.In certain further embodiments the minimal furin cleavage site isrepresented by the consensus amino acid sequence R/Y-x-x-R and/orR-x-x-R.

In certain embodiments of Embodiment Set #13, the cell-targetingmolecule comprises a molecular moiety located carboxy-terminal to thecarboxy-terminus of the Shiga toxin A1 fragment region or Shiga toxin A1fragment derived region.

In certain embodiments of Embodiment Set #13, the binding regionsterically covers the carboxy-terminus of the A1 fragment region orShiga toxin A1 fragment derived region.

In certain embodiments of Embodiment Set #13, the molecular moietysterically covers the carboxy-terminus of the A1 fragment region orShiga toxin A1 fragment derived region. In certain further embodiments,the molecular moiety comprises the binding region.

In certain embodiments of Embodiment Set #13, the cell-targetingmolecule of the present invention comprises a binding region and/ormolecular moiety located carboxy-terminal to the carboxy-terminus of theShiga toxin A1 fragment region or Shiga toxin A1 fragment derivedregion. In certain further embodiments, the mass of the binding regionand/or molecular moiety is at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, orgreater.

In certain embodiments of Embodiment Set #13, the cell-targetingmolecule comprises a binding region with a mass of at least 4.5 kDa, 6,kDa, 9 kDa, 12 kDa, kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa,100 kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein(e.g., cytotoxicity and/or intracellular routing).

In certain embodiments of Embodiment Set #13, the binding region iscomprised within a relatively large, molecular moiety comprising suchas, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein.

In certain embodiments of Embodiment Set #13, the disruptedfurin-cleavage motif comprises an amino acid residue substitution in thefurin-cleavage motif relative to a wild-type Shiga toxin A Subunit. Incertain further embodiments, the substitution of the amino acid residuein the furin-cleavage motif is of an arginine residue with anon-positively charged, amino acid residue selected from the groupconsisting of: alanine, glycine, proline, serine, threonine, aspartate,asparagine, glutamate, glutamine, cysteine, isoleucine, leucine,methionine, valine, phenylalanine, tryptophan, and tyrosine. In certainembodiments, the substitution of the amino acid residue in thefurin-cleavage motif is of an arginine residue with a histidine.

In certain embodiments of Embodiment Set #13, the cell-targetingmolecule is capable when introduced to cells of exhibiting cytotoxicitycomparable to the cytotoxicity of a seventh cell-targeting moleculeconsisting of the cell-targeting molecule except for all of its Shigatoxin effector polypeptide component(s) each comprise a wild-type Shigatoxin A1 fragment and/or wild-type Shiga toxin furin-cleavage site atthe carboxy terminus of its A1 fragment region and/or Shiga toxin A1fragment derived region. In certain further embodiments, thecell-targeting molecule of the present invention is capable whenintroduced to cells of exhibiting cytotoxicity that is in a range offrom 0.1-fold, 0.5-fold, or 0.75-fold to 1.2-fold, 1.5-fold, 1.75-fold,2-fold, 3-fold, 4-fold, or 5-fold of the cytotoxicity exhibited by theseventh cell-targeting molecule.

In certain embodiments of Embodiment Set #13, the cell-targetingmolecule is capable when introduced to a chordate of exhibitingimproved, in vivo tolerability compared to in vivo tolerability of theseventh cell-targeting molecule. For embodiments, wherein the molecularmoiety is not toxic and the molecular moiety comprises the bindingregion, the cell-targeting molecule is capable when introduced to achordate of exhibiting improved, in vivo tolerability compared to invivo tolerability of the seventh cell-targeting molecule only when allthe binding regions of the cell-targeting molecule are associated orlinked, either directly or indirectly, to a Shiga toxin effectorpolypeptide at a position carboxy-terminal to the carboxy-terminus ofthe Shiga toxin A1 fragment region and/or Shiga toxin A1 fragmentderived region of that Shiga toxin effector polypeptide.

In certain embodiments of Embodiment Set #13, the cell-targetingmolecule is de-immunized due to the embedded or inserted, heterologousepitope. In certain further embodiments, the cell-targeting molecule iscapable of exhibiting less relative antigenicity and/or relativeimmunogenicity as compared to a reference molecule, such as, e.g., aneighth cell-targeting molecule consisting of the cell-targeting moleculeexcept for it lacks one or more embedded or inserted epitopes present inthe cell targeting molecule.

In certain embodiments of Embodiment Set #13, the cell-targetingmolecule is de-immunized due to the furin-cleavage motif disruption. Incertain further embodiments, the cell-targeting molecule is capable ofexhibiting less relative antigenicity and/or relative immunogenicity ascompared to a ninth cell-targeting molecule consisting of thecell-targeting molecule except for the furin-cleavage motif is wild-typeand/or all the Shiga toxin effector polypeptide components consist of awild-type Shiga toxin A1 fragment.

Embodiment Set #14—Multivalent Cell-Targeting Molecules Comprising aShiga Toxin Effector Polypeptide at or Proximal to an Amino-Terminus andWherein the Shiga Toxin Effector Polypeptide Comprises an Embedded orInserted, Heterologous, T-Cell Epitope

The present invention provides multivalent cell-targeting molecules,each comprising (i) two or more binding regions, each capable ofspecifically binding an extracellular part of the same targetbiomolecule; (ii) a Shiga toxin effector polypeptide comprising aninserted or embedded, heterologous epitope; wherein the Shiga toxineffector polypeptide is at or proximal to an amino-terminus of apolypeptide. In certain embodiments, the cell-targeting molecule of thepresent invention comprises (i) a binding region capable of specificallybinding an extracellular target biomolecule, (ii) a polypeptidecomponent, and (iii) a Shiga toxin effector polypeptide comprising aninserted or embedded, heterologous epitope: wherein the Shiga toxineffector polypeptide is at or proximal to an amino-terminus of thepolypeptide component of the cell-targeting molecule. In certain furtherembodiments, the binding region and Shiga toxin effector polypeptide arephysically arranged or oriented within the cell-targeting molecule suchthat the binding region is not located proximally to the amino-terminusof the Shiga toxin effector polypeptide. In certain further embodiments,the binding region is located within the cell-targeting molecule moreproximal to the carboxy-terminus of the Shiga toxin effector polypeptidethan to the amino-terminus of the Shiga toxin effector polypeptide. Incertain further embodiments, the binding region is not locatedproximally to an amino-terminus of the cell-targeting molecule relativeto the Shiga toxin effector polypeptide. For certain furtherembodiments, the Shiga toxin effector polypeptide is capable ofexhibiting at least one Shiga toxin effector function, such as, e.g.,directing intracellular routing to the endoplasmic reticulum and/orcytosol of a cell in which the polypeptide is present, inhibiting aribosome function, enzymatically inactivating a ribosome, causingcytostasis, and/or causing cytotoxicity. In certain further embodiments,the heterologous, T-cell epitope is a CD8+ T-cell epitope, such as,e.g., with regard to a human immune system. For certain furtherembodiments, the heterologous, T-cell epitope is capable of beingpresented by a MHC class I molecule of a cell. In certain furtherembodiments, the cell-targeting molecule of the present invention iscapable of one or more the following: entering a cell, inhibiting aribosome function, causing cytostasis, causing cell death, and/ordelivering the embedded or inserted, heterologous, T-cell epitope to aMHC class I molecule for presentation on a cellular surface.

In certain embodiments of Embodiment Set #14, the cell-targetingmolecule of the present invention is capable when introduced to cells ofexhibiting cytotoxicity that is greater than that of a tenthcell-targeting molecule having an amino-terminus and comprising thebinding region and the Shiga toxin effector polypeptide region which isnot positioned at or proximal to the amino-terminus of the tenthcell-targeting molecule. In certain further embodiments, thecell-targeting molecule of the present invention is capable ofexhibiting a cytotoxicity with better optimized, cytotoxic potency, suchas, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity ascompared to the tenth cell-targeting molecule. In certain furtherembodiments, the cytotoxicity of the cell-targeting molecule of thepresent invention to a population of target positive cells is 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater thanthe cytotoxicity of the tenth cell-targeting molecule to a secondpopulation of target positive cells as assayed by CD₅₀ values.

For certain embodiments of Embodiment Set #14, the cell-targetingmolecule of the present invention is capable of delivering an embeddedor inserted, heterologous, CD8+ T-cell epitope to a MHC class Ipresentation pathway of a cell for cell-surface presentation of theepitope bound by a MHC class I molecule.

In certain embodiments of Embodiment Set #14, the cell-targetingmolecule is de-immunized due to the embedded or inserted, heterologousepitope. In certain further embodiments, the cell-targeting molecule iscapable of exhibiting less relative antigenicity and/or relativeimmunogenicity as compared to a reference molecule, such as, e.g., aneleventh cell-targeting molecule consisting of the cell-targetingmolecule except for it lacks one or more embedded or inserted epitopespresent in the cell targeting molecule.

For certain further embodiments of Embodiment Set #14, thecell-targeting molecule of the present invention is not cytotoxic and iscapable when introduced to cells of exhibiting a greater subcellularrouting efficiency from an extracellular space to a subcellularcompartment of an endoplasmic reticulum and/or cytosol as compared tothe cytotoxicity of a reference molecule, such as, e.g., the tenthcell-targeting molecule.

Embodiment Set #15—Multivalent Cell-Targeting Molecule Comprising aDe-Immunized Shiga Toxin Effector Polypeptide Comprising a Disrupted,Furin-Cleavage Motif

The present invention provides multivalent cell-targeting molecules,each comprising (i) two or more binding regions, each capable ofspecifically binding an extracellular part of the same targetbiomolecule, and (ii) a de-immunized. Shiga toxin effector polypeptidecomprising a disrupted furin-cleavage motif. In certain embodiments, thecell-targeting molecule of the present invention comprises (i) a bindingregion capable of specifically binding an extracellular targetbiomolecule and (ii) a de-immunized, Shiga toxin effector polypeptidecomprising (a) a Shiga toxin A1 fragment derived region having a carboxyterminus, (b) a disrupted furin-cleavage motif at the carboxy-terminusof the A1 fragment region, and (c) at least one disrupted, endogenous,B-cell and/or CD4+ T-cell epitope and/or epitope region. For certainfurther embodiments, the Shiga toxin effector polypeptide is capable ofexhibiting at least one Shiga toxin effector function, such as, e.g.,directing intracellular routing to the endoplasmic reticulum and/orcytosol of a cell in which the polypeptide is present, inhibiting aribosome function, enzymatically inactivating a ribosome, causingcytostasis, and/or causing cytotoxicity. In certain further embodiments,the cell-targeting molecule of the present invention is capable of oneor more the following: entering a cell, inhibiting a ribosome function,causing cytostasis, and/or causing cell death. For certain furtherembodiments, the cell-targeting molecule is capable when introduced tocells of exhibiting a cytotoxicity comparable or better than a referencemolecule, such as, e.g., a second cell-targeting molecule consisting ofthe cell-targeting molecule except for all of its Shiga toxin effectorpolypeptide components comprise a wild-type Shiga toxin furin-cleavagesite at the carboxy terminus of its A1 fragment region.

In certain embodiments of Embodiment Set #15, the Shiga toxin effectorpolypeptide comprises a mutation, relative to a wild-type Shiga toxin ASubunit, in the B-cell and/or T-cell epitope region selected from thegroup of natively positioned Shiga toxin A Subunit regions consisting of1-15 of any one of SEQ ID NOs: 1-2 and 4-6; 3-14 of any one of SEQ IDNOs: 3 and 7-18; 26-37 of any one of SEQ ID NOs: 3 and 7-18; 27-37 ofany one of SEQ ID NOs: 1-2 and 4-6; 39-48 of any one of SEQ ID NOs: 1-2and 4-6; 42-48 of any one of SEQ ID NOs: 3 and 7-18; and 53-66 of anyone of SEQ ID NOs: 1-18; 94-115 of any one of SEQ ID NOs: 1-18; 141-153of any one of SEQ ID NOs: 1-2 and 4-6; 140-156 of any one of SEQ ID NOs:3 and 7-18; 179-190 of any one of SEQ ID NOs: 1-2 and 4-6; 179-191 ofany one of SEQ ID NOs: 3 and 7-18; 204 of SEQ ID NO:3; 205 of any one ofSEQ ID NOs: 1-2 and 4-6; and 210-218 of any one of SEQ ID NOs: 3 and7-18; 240-260 of any one of SEQ ID NOs: 3 and 7-18; 243-257 of any oneof SEQ ID NOs: 1-2 and 4-6; 254-268 of any one of SEQ ID NOs: 1-2 and4-6; 262-278 of any one of SEQ ID NOs: 3 and 7-18; 281-297 of any one ofSEQ ID NOs: 3 and 7-18; and 285-293 of any one of SEQ ID NOs: 1-2 and4-6; or the equivalent region in a Shiga toxin A Subunit or derivativethereof. In certain further embodiments, there is no disruption which isa carboxy-terminal truncation of amino acid residues that overlap withpart or all of at least one disrupted, endogenous, B-cell and/or T-cellepitope and/or epitope region.

In certain embodiments of Embodiment Set #15, the disruptedfurin-cleavage motif comprises one or more mutations, relative to awild-type Shiga toxin A Subunit, the mutation altering at least oneamino acid residue in a region natively positioned at 248-251 of the ASubunit of Shiga toxin (SEQ ID NOs: 1-2 and 4-6), or at 247-250 of the ASubunit of Shiga-like toxin 2 (SEQ ID NOs: 3 and 7-18), or theequivalent region in a Shiga toxin A Subunit or derivative thereof. Incertain further embodiments, the disrupted furin-cleavage motifcomprises one or more mutations, relative to a wild-type Shiga toxin ASubunit, in a minimal furin cleavage site of the furin-cleavage motif.In certain further embodiments the minimal furin cleavage site isrepresented by the consensus amino acid sequence R/Y-x-x-R and/orR-x-x-R.

In certain embodiments of Embodiment Set #15, the cell-targetingmolecule comprises a molecular moiety located carboxy-terminal to thecarboxy-terminus of the Shiga toxin A1 fragment region.

In certain embodiments of Embodiment Set #15, the binding regionsterically covers the carboxy-terminus of the A1 fragment region.

In certain embodiments of Embodiment Set #15, the molecular moietysterically covers the carboxy-terminus of the A1 fragment region. Incertain further embodiments, the molecular moiety comprises the bindingregion.

In certain embodiments of Embodiment Set #15, the cell-targetingmolecule of the present invention comprises a binding region and/ormolecular moiety located carboxy-terminal to the carboxy-terminus of theShiga toxin A1 fragment region. In certain further embodiments, the massof the binding region and/or molecular moiety is at least 4.5 kDa, 6,kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, kDa, 41 kDa, 50 kDa,100 kDa, or greater.

In certain embodiments of Embodiment Set #15, the cell-targetingmolecule comprises a binding region with a mass of at least 4.5 kDa, 6,kDa, 9 kDa, 12 kDa, kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa,100 kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein(e.g., cytotoxicity and/or intracellular routing).

In certain embodiments of Embodiment Set #15, the binding region iscomprised within a relatively large, molecular moiety comprising suchas, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein.

In certain embodiments of Embodiment Set #15, the disruptedfurin-cleavage motif comprises an amino acid residue substitution in thefurin-cleavage motif relative to a wild-type Shiga toxin A Subunit. Incertain further embodiments, the substitution of the amino acid residuein the furin-cleavage motif is of an arginine residue with anon-positively charged, amino acid residue selected from the groupconsisting of: alanine, glycine, proline, serine, threonine, aspartate,asparagine, glutamate, glutamine, cysteine, isoleucine, leucine,methionine, valine, phenylalanine, tryptophan, and tyrosine. In certainembodiments, the substitution of the amino acid residue in thefurin-cleavage motif is of an arginine residue with a histidine.

In certain embodiments of Embodiment Set #15, the cell-targetingmolecule is capable when introduced to cells of exhibiting cytotoxicitycomparable to the cytotoxicity of a reference molecule, such as, e.g., atwelfth cell-targeting molecule consisting of the cell-targetingmolecule except for all of its Shiga toxin effector polypeptidecomponent(s) each comprise a wild-type Shiga toxin A1 fragment and/orwild-type Shiga toxin furin-cleavage site at the carboxy terminus of itsA1 fragment region. In certain further embodiments, the cell-targetingmolecule of the present invention is capable when introduced to cells ofexhibiting cytotoxicity that is in a range of from 0.1-fold, 0.5-fold,or 0.75-fold to 1.2-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold,or 5-fold of the cytotoxicity exhibited by the twelfth cell-targetingmolecule.

In certain embodiments of Embodiment Set #15, the cell-targetingmolecule is capable when introduced to a chordate of exhibitingimproved, in vivo tolerability compared to in vivo tolerability of thetwelfth cell-targeting molecule.

In certain embodiments of Embodiment Set #15, the cell-targetingmolecule is de-immunized due to the furin-cleavage motif disruption. Incertain further embodiments, the cell-targeting molecule is capable ofexhibiting less relative antigenicity and/or relative immunogenicity ascompared to a reference cell-targeting molecule consisting of thecell-targeting molecule except for the furin-cleavage motif is wild-typeand/or all the Shiga toxin effector polypeptide components consist of awild-type Shiga toxin A1 fragment, such as, e.g., the twelfthcell-targeting molecule.

In certain embodiments of Embodiment Set #15, the cell-targetingmolecule of the present invention comprises two proteins selected fromany one of the polypeptides shown in any one of SEQ ID NOs: 252-255,259-271, 274-278 and 288-748, and which optionally comprises anamino-terminal methionine residue.

Embodiment Set #16—Multivalent Cell-Targeting Molecule Comprising aCarboxy-Terminal Endoplasmic Reticulum Retention/Retrieval Signal Motifand a De-Immunized Shiga Toxin Effector Polypeptide

The present invention provides multivalent cell-targeting molecules,each comprising (i) two or more binding regions, each capable ofspecifically binding an extracellular part of the same targetbiomolecule; (ii) a de-immunized, Shiga toxin effector polypeptide, and(iii) a carboxy-terminal, endoplasmic reticulum retention/retrievalsignal motif. In certain embodiments, the cell-targeting molecule of thepresent invention comprises (i) a binding region capable of specificallybinding an extracellular target biomolecule; (ii) a de-immunized, Shigatoxin effector polypeptide comprising at least one disrupted,endogenous. B-cell and/or CD4+ T-cell epitope and/or epitope region, and(iii) a carboxy-terminal, endoplasmic reticulum retention/retrievalsignal motif of a member of the KDEL family. For certain furtherembodiments, the Shiga toxin effector polypeptide is capable ofexhibiting at least one Shiga toxin effector function, such as, e.g.,directing intracellular routing to the endoplasmic reticulum and/orcytosol of a cell in which the polypeptide is present, inhibiting aribosome function, enzymatically inactivating a ribosome, causingcytostasis, and/or causing cytotoxicity. In certain further embodiments,the cell-targeting molecule of the present invention is capable of oneor more the following: entering a cell, inhibiting a ribosome function,causing cytostasis, and/or causing cell death.

In certain embodiments of Embodiment Set #16, the carboxy-terminalendoplasmic reticulum retention/retrieval signal motif is selected fromthe group consisting of: KDEL, HDEF. HDEL, RDEF, RDEL, WDEL, YDEL, HEEF,HEEL, KEEL, REEL, KAEL, KCEL, KFEL. KGEL, KHEL, KLEL, KNEL, KQEL, KREL,KSEL, KVEL. KWEL. KYEL, KEDL, KIEL, DKEL, FDEL, KDEF. KKEL, HADL, HAEL,HIEL, HNEL, HTEL, KTEL, HVEL, NDEL, QDEL, REDL, RNEL, RTDL, RTEL, SDEL.TDEL. SKEL, STEL, and EDEL.

In certain embodiments of Embodiment Set #16, the Shiga toxin effectorpolypeptide comprises a mutation, relative to a wild-type Shiga toxin ASubunit, in the B-cell and/or T-cell epitope region selected from thegroup of natively positioned Shiga toxin A Subunit regions consistingof: 1-15 of any one of SEQ ID NOs: 1-2 and 4-6; 3-14 of any one of SEQID NOs: 3 and 7-18; 26-37 of any one of SEQ ID NOs: 3 and 7-18; 27-37 ofany one of SEQ ID NOs: 1-2 and 4-6; 39-48 of any one of SEQ ID NOs: 1-2and 4-6; 42-48 of any one of SEQ ID NOs: 3 and 7-18; and 53-66 of anyone of SEQ ID NOs: 1-18; 94-115 of any one of SEQ ID NOs: 1-18; 141-153of any one of SEQ ID NOs: 1-2 and 4-6; 140-156 of any one of SEQ ID NOs:3 and 7-18; 179-190 of any one of SEQ ID NOs: 1-2 and 4-6; 179-191 ofany one of SEQ ID NOs: 3 and 7-18; 204 of SEQ ID NO:3; 205 of any one ofSEQ ID NOs: 1-2 and 4-6; and 210-218 of any one of SEQ ID NOs: 3 and7-18; 240-260 of any one of SEQ ID NOs: 3 and 7-18; 243-257 of any oneof SEQ ID NOs: 1-2 and 4-6; 254-268 of any one of SEQ ID NOs: 1-2 and4-6; 262-278 of any one of SEQ ID NOs: 3 and 7-18; 281-297 of any one ofSEQ ID NOs: 3 and 7-18; and 285-293 of any one of SEQ ID NOs: 1-2 and4-6; or the equivalent region in a Shiga toxin A Subunit or derivativethereof. In certain further embodiments, there is no disruption which isa carboxy-terminal truncation of amino acid residues that overlap withpart or all of at least one disrupted, endogenous, B-cell and/or T-cellepitope and/or epitope region.

In certain embodiments of Embodiment Set #16, the cell-targetingmolecule of the present invention is capable when introduced to cells ofexhibiting cytotoxicity that is greater than that of a thirteenthcell-targeting molecule consisting of the cell-targeting molecule exceptfor it does not comprise any carboxy-terminal, endoplasmic reticulumretention/retrieval signal motif of the KDEL family. In certain furtherembodiments, the cell-targeting molecule of the present invention iscapable of exhibiting a cytotoxicity with better optimized, cytotoxicpotency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greatercytotoxicity as compared to the thirteenth cell-targeting molecule. Incertain further embodiments, the cytotoxicity of the cell-targetingmolecule of the present invention to a population of target positivecells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-foldor greater than the cytotoxicity of the thirteenth cell-targetingmolecule to a second population of target positive cells as assayed byCD₅₀ values.

For certain further embodiments of Embodiment Set #16, thecell-targeting molecule of the present invention is not cytotoxic and iscapable when introduced to cells of exhibiting a greater subcellularrouting efficiency from an extracellular space to a subcellularcompartment of an endoplasmic reticulum and/or cytosol as compared tothe cytotoxicity of a reference molecule, such as, e.g., the thirteenthcell-targeting molecule.

Embodiment Set #17—Multivalent Cell-Targeting Molecule Comprising aDe-Immunized Shiga Toxin Effector Polypeptide at or Proximal to anAmino-Terminus of the Cell Targeting Molecule

The present invention provides multivalent cell-targeting molecules,each comprising (i) two or more binding regions, each capable ofspecifically binding an extracellular part of the same targetbiomolecule, (ii) a de-immunized, Shiga toxin effector polypeptide;wherein the Shiga toxin effector polypeptide is at or proximal to anamino-terminus. In certain embodiments, the cell-targeting molecule ofthe present invention comprises (i) a binding region capable ofspecifically binding an extracellular target biomolecule; (ii)polypeptide component; and (iii) a de-immunized, Shiga toxin effectorpolypeptide comprising at least one disrupted, endogenous, B-cell and/orCD4+ T-cell epitope and/or epitope region; wherein the Shiga toxineffector polypeptide is at or proximal to an amino-terminus of thepolypeptide component of the cell-targeting molecule. In certain furtherembodiments, the binding region and Shiga toxin effector polypeptide arephysically arranged or oriented within the cell-targeting molecule suchthat the binding region is not located proximally to the amino-terminusof the Shiga toxin effector polypeptide. In certain further embodiments,the binding region is located within the cell-targeting molecule moreproximal to the carboxy-terminus of the Shiga toxin effector polypeptidethan to the amino-terminus of the Shiga toxin effector polypeptide. Incertain further embodiments, the binding region is not locatedproximally to an amino-terminus of the cell-targeting molecule relativeto the Shiga toxin effector polypeptide. For certain furtherembodiments, the Shiga toxin effector polypeptide is capable ofexhibiting at least one Shiga toxin effector function, such as, e.g.,directing intracellular routing to the endoplasmic reticulum and/orcytosol of a cell in which the polypeptide is present, inhibiting aribosome function, enzymatically inactivating a ribosome, causingcytostasis, and/or causing cytotoxicity. In certain further embodiments,the cell-targeting molecule of the present invention is capable of oneor more the following: entering a cell, inhibiting a ribosome function,causing cytostasis, and/or causing cell death.

In certain embodiments of Embodiment Set #17, the Shiga toxin effectorpolypeptide comprises a mutation, relative to a wild-type Shiga toxin ASubunit, in the B-cell and/or T-cell epitope region selected from thegroup of natively positioned Shiga toxin A Subunit regions consistingof: 1-15 of any one of SEQ ID NOs: 1-2 and 4-6; 3-14 of any one of SEQID NOs: 3 and 7-18; 26-37 of any one of SEQ ID NOs: 3 and 7-18; 27-37 ofany one of SEQ ID NOs: 1-2 and 4-6; 39-48 of any one of SEQ ID NOs: 1-2and 4-6; 42-48 of any one of SEQ ID NOs: 3 and 7-18; and 53-66 of anyone of SEQ ID NOs: 1-18; 94-115 of any one of SEQ ID NOs: 1-18; 141-153of any one of SEQ ID NOs: 1-2 and 4-6; 140-156 of any one of SEQ ID NOs:3 and 7-18; 179-190 of any one of SEQ ID NOs: 1-2 and 4-6; 179-191 ofany one of SEQ ID NOs: 3 and 7-18; 204 of SEQ ID NO:3; 205 of any one ofSEQ ID NOs: 1-2 and 4-6; and 210-218 of any one of SEQ ID NOs: 3 and7-18; 240-260 of any one of SEQ ID NOs: 3 and 7-18; 243-257 of any oneof SEQ ID NOs: 1-2 and 4-6; 254-268 of any one of SEQ ID NOs: 1-2 and4-6; 262-278 of any one of SEQ ID NOs: 3 and 7-18; 281-297 of any one ofSEQ ID NOs: 3 and 7-18; and 285-293 of any one of SEQ ID NOs: 1-2 and4-6; or the equivalent region in a Shiga toxin A Subunit or derivativethereof. In certain further embodiments, there is no disruption which isa carboxy-terminal truncation of amino acid residues that overlap withpart or all of at least one disrupted, endogenous, B-cell and/or T-cellepitope and/or epitope region.

In certain embodiments of Embodiment Set #17, the cell-targetingmolecule of the present invention is capable when introduced to cells ofexhibiting cytotoxicity that is greater than that of a fourteenthcell-targeting molecule having an amino-terminus and comprising thebinding region and the Shiga toxin effector polypeptide region which isnot positioned at or proximal to the amino-terminus of the fourteenthcell-targeting molecule. In certain further embodiments, thecell-targeting molecule of the present invention is capable ofexhibiting a cytotoxicity with better optimized, cytotoxic potency, suchas, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity ascompared to the fourteenth cell-targeting molecule. In certain furtherembodiments, the cytotoxicity of the cell-targeting molecule of thepresent invention to a population of target positive cells is 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater thanthe cytotoxicity of the fourteenth cell-targeting molecule to a secondpopulation of target positive cells as assayed by CD₅₀ values.

For certain further embodiments of Embodiment Set #17, thecell-targeting molecule of the present invention is not cytotoxic and iscapable when introduced to cells of exhibiting a greater subcellularrouting efficiency from an extracellular space to a subcellularcompartment of an endoplasmic reticulum and/or cytosol as compared tothe cytotoxicity of a reference molecule, such as, e.g., the fourteenthcell-targeting molecule.

In certain embodiments of Embodiment Set #17, the cell-targetingmolecule of the present invention comprises two proteins selected fromany one of the polypeptides shown in any one of SEQ ID NOs: 252-255,259-271, 274-278 and 288-748, and which optionally comprises anamino-terminal methionine residue.

Embodiment Set #18—Multivalent Cell-Targeting Molecule Comprising aCarboxy-Terminal Endoplasmic Reticulum Retention/Retrieval Signal Motifand a Shiga Toxin Effector Polypeptide Comprising a Disrupted,Furin-Cleavage Motif

The present invention provides multivalent cell-targeting molecules,each comprising (i) two or more binding regions, each capable ofspecifically binding an extracellular part of the same targetbiomolecule; (ii) a Shiga toxin effector polypeptide comprising adisrupted furin-cleavage motif; and (iii) a carboxy-terminal endoplasmicreticulum retention/retrieval signal motif. The present inventionprovides cell-targeting molecules, each comprising (i) a binding regioncapable of specifically binding an extracellular target biomolecule;(ii) a Shiga toxin effector polypeptide comprising a disruptedfurin-cleavage motif; and (iii) a carboxy-terminal, endoplasmicreticulum retention/retrieval signal motif of a member of the KDELfamily. For certain further embodiments, the Shiga toxin effectorpolypeptide is capable of exhibiting at least one Shiga toxin effectorfunction, such as, e.g., directing intracellular routing to theendoplasmic reticulum and/or cytosol of a cell in which the polypeptideis present, inhibiting a ribosome function, enzymatically inactivating aribosome, causing cytostasis, and/or causing cytotoxicity. In certainfurther embodiments, the cell-targeting molecule of the presentinvention is capable of one or more the following: entering a cell,inhibiting a ribosome function, causing cytostasis, and/or causing celldeath. For certain further embodiments, the cell-targeting molecule iscapable when introduced to cells of exhibiting a cytotoxicity comparableor better than a reference molecule, such as, e.g., a secondcell-targeting molecule consisting of the cell-targeting molecule exceptfor all of its Shiga toxin effector polypeptide components comprise awild-type Shiga toxin furin-cleavage site at the carboxy terminus of itsA1 fragment region.

In certain embodiments of Embodiment Set #18, the disruptedfurin-cleavage motif comprises one or more mutations, relative to awild-type Shiga toxin A Subunit, the mutation altering at least oneamino acid residue in a region natively positioned at 248-251 of the ASubunit of Shiga toxin (SEQ ID NOs: 1-2 and 4-6), or at 247-250 of the ASubunit of Shiga-like toxin 2 (SEQ ID NOs: 3 and 7-18), or theequivalent region in a Shiga toxin A Subunit or derivative thereof. Incertain further embodiments, the disrupted furin-cleavage motifcomprises one or more mutations, relative to a wild-type Shiga toxin ASubunit, in a minimal furin cleavage site of the furin-cleavage motif.In certain further embodiments the minimal furin cleavage site isrepresented by the consensus amino acid sequence R/Y-x-x-R and/orR-x-x-R.

In certain embodiments of Embodiment Set #18, the cell-targetingmolecule comprises a molecular moiety located carboxy-terminal to thecarboxy-terminus of the Shiga toxin A1 fragment region.

In certain embodiments of Embodiment Set #18, the binding regionsterically covers the carboxy-terminus of the A1 fragment region.

In certain embodiments of Embodiment Set #18, the molecular moietysterically covers the carboxy-terminus of the A1 fragment region. Incertain further embodiments, the molecular moiety comprises the bindingregion.

In certain embodiments of Embodiment Set #18, the cell-targetingmolecule of the present invention comprises a binding region and/ormolecular moiety located carboxy-terminal to the carboxy-terminus of theShiga toxin A1 fragment region. In certain further embodiments, the massof the binding region and/or molecular moiety is at least 4.5 kDa, 6,kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, kDa, 41 kDa, 50 kDa,100 kDa, or greater.

In certain embodiments of Embodiment Set #18, the cell-targetingmolecule comprises a binding region with a mass of at least 4.5 kDa, 6,kDa, 9 kDa, 12 kDa, kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa,100 kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein(e.g., cytotoxicity and/or intracellular routing).

In certain embodiments of Embodiment Set #18, the binding region iscomprised within a relatively large, molecular moiety comprising suchas, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein.

In certain embodiments of Embodiment Set #18, the disruptedfurin-cleavage motif comprises an amino acid residue substitution in thefurin-cleavage motif relative to a wild-type Shiga toxin A Subunit. Incertain further embodiments, the substitution of the amino acid residuein the furin-cleavage motif is of an arginine residue with anon-positively charged, amino acid residue selected from the groupconsisting of alanine, glycine, proline, serine, threonine, aspartate,asparagine, glutamate, glutamine, cysteine, isoleucine, leucine,methionine, valine, phenylalanine, tryptophan, and tyrosine. In certainembodiments, the substitution of the amino acid residue in thefurin-cleavage motif is of an arginine residue with a histidine.

In certain embodiments of Embodiment Set #18, the cell-targetingmolecule of the present invention is capable when introduced to cells ofexhibiting cytotoxicity that is greater than that of a fifteenthcell-targeting molecule consisting of the cell-targeting molecule exceptfor it does not comprise any carboxy-terminal, endoplasmic reticulumretention/retrieval signal motif of the KDEL family. In certain furtherembodiments, the cell-targeting molecule of the present invention iscapable of exhibiting a cytotoxicity with better optimized, cytotoxicpotency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greatercytotoxicity as compared to the fifteenth cell-targeting molecule. Incertain further embodiments, the cytotoxicity of the cell-targetingmolecule of the present invention to a population of target positivecells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-foldor greater than the cytotoxicity of the fifteenth cell-targetingmolecule to a second population of target positive cells as assayed byCD₅₀ values.

In certain embodiments of Embodiment Set #18, the cell-targetingmolecule is capable when introduced to a chordate of exhibitingimproved, in vivo tolerability compared to in vivo tolerability of asixteenth cell-targeting molecule consisting of the cell-targetingmolecule except for all of its Shiga toxin effector polypeptidecomponent(s) each comprise a wild-type Shiga toxin A1 fragment and/orwild-type Shiga toxin furin-cleavage site at the carboxy terminus of itsA1 fragment region.

In certain embodiments of Embodiment Set #18, the cell-targetingmolecule is de-immunized due to the furin-cleavage motif disruption. Incertain further embodiments, the cell-targeting molecule is capable ofexhibiting less relative antigenicity and/or relative immunogenicity ascompared to a reference cell-targeting molecule consisting of thecell-targeting molecule except for the furin-cleavage motif is wild-typeand/or all the Shiga toxin effector polypeptide components consist of awild-type Shiga toxin A1 fragment, such as, e.g., the sixteenthcell-targeting molecule.

For certain further embodiments of Embodiment Set #18, thecell-targeting molecule of the present invention is not cytotoxic and iscapable when introduced to cells of exhibiting a greater subcellularrouting efficiency from an extracellular space to a subcellularcompartment of an endoplasmic reticulum and/or cytosol as compared tothe cytotoxicity of a reference molecule, such as, e.g., the fifteenthcell-targeting molecule.

Embodiment Set #19—Multivalent Cell-Targeting Molecule Comprising aFurin-Cleavage Resistant Shiga Toxin Effector Polypeptide at or Proximalto an Amino-Terminus of the Cell Targeting Molecule

The present invention provides multivalent cell-targeting molecules,each comprising (i) two or more binding regions, each capable ofspecifically binding an extracellular part of the same targetbiomolecule, and (ii) a Shiga toxin effector polypeptide comprising adisrupted furin-cleavage motif at the carboxy-terminus of its Shigatoxin A1 fragment region; wherein the amino-terminus of the Shiga toxineffector polypeptide is at and/or proximal to an amino-terminus of apolypeptide component of the cell-targeting molecule. In certainembodiments, the cell-targeting molecule of the present inventioncomprises (i) a binding region capable of specifically binding anextracellular target biomolecule, (ii) a Shiga toxin effectorpolypeptide having an amino-terminus and a Shiga toxin A1 fragmentderived region having a carboxy terminus, and (iii) a disruptedfurin-cleavage motif at the carboxy-terminus of the A1 fragment region;wherein the binding region is not located proximally to theamino-terminus of the cell-targeting molecule relative to the Shigatoxin effector polypeptide. In certain further embodiments, the bindingregion and Shiga toxin effector polypeptide are physically arranged ororiented within the cell-targeting molecule such that the binding regionis not located proximally to the amino-terminus of the Shiga toxineffector polypeptide. In certain further embodiments, the binding regionis located within the cell-targeting molecule more proximal to thecarboxy-terminus of the Shiga toxin effector polypeptide than to theamino-terminus of the Shiga toxin effector polypeptide. In certainfurther embodiments, the binding region is not located proximally to anamino-terminus of the cell-targeting molecule relative to the Shigatoxin effector polypeptide. For certain further embodiments, the Shigatoxin effector polypeptide is capable of exhibiting at least one Shigatoxin effector function, such as, e.g., directing intracellular routingto the endoplasmic reticulum and/or cytosol of a cell in which thepolypeptide is present, inhibiting a ribosome function, enzymaticallyinactivating a ribosome, causing cytostasis, and/or causingcytotoxicity. In certain further embodiments, the cell-targetingmolecule of the present invention is capable of one or more thefollowing: entering a cell, inhibiting a ribosome function, causingcytostasis, and/or causing cell death. For certain further embodiments,the cell-targeting molecule is capable when introduced to cells ofexhibiting a cytotoxicity comparable or better than a referencemolecule, such as, e.g., a seventeenth cell-targeting moleculeconsisting of the cell-targeting molecule except for all of its Shigatoxin effector polypeptide components comprise a wild-type Shiga toxinfurin-cleavage site at the carboxy terminus of its A1 fragment region.

In certain embodiments of Embodiment Set #19, the disruptedfurin-cleavage motif comprises one or more mutations, relative to awild-type Shiga toxin A Subunit, the mutation altering at least oneamino acid residue in a region natively positioned at 248-251 of the ASubunit of Shiga toxin (SEQ ID NOs: 1-2 and 4-6), or at 247-250 of the ASubunit of Shiga-like toxin 2 (SEQ ID NOs: 3 and 7-18), or theequivalent region in a Shiga toxin A Subunit or derivative thereof. Incertain further embodiments, the disrupted furin-cleavage motifcomprises one or more mutations, relative to a wild-type Shiga toxin ASubunit, in a minimal furin cleavage site of the furin-cleavage motif.In certain further embodiments the minimal furin cleavage site isrepresented by the consensus amino acid sequence R/Y-x-x-R and/orR-x-x-R.

In certain embodiments of Embodiment Set #19, the cell-targetingmolecule comprises a molecular moiety located carboxy-terminal to thecarboxy-terminus of the Shiga toxin A1 fragment region.

In certain embodiments of Embodiment Set #19, the binding regionsterically covers the carboxy-terminus of the A1 fragment region.

In certain embodiments of Embodiment Set #19, the molecular moietysterically covers the carboxy-terminus of the A1 fragment region. Incertain further embodiments, the molecular moiety comprises the bindingregion.

In certain embodiments of Embodiment Set #19, the cell-targetingmolecule of the present invention comprises a binding region and/ormolecular moiety located carboxy-terminal to the carboxy-terminus of theShiga toxin A1 fragment region. In certain further embodiments, the massof the binding region and/or molecular moiety is at least 4.5 kDa, 6,kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, kDa, 41 kDa, 50 kDa,100 kDa, or greater.

In certain embodiments of Embodiment Set #19, the cell-targetingmolecule comprises a binding region with a mass of at least 4.5 kDa, 6,kDa, 9 kDa, 12 kDa, kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa,100 kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein(e.g., cytotoxicity and/or intracellular routing).

In certain embodiments of Embodiment Set #19, the binding region iscomprised within a relatively large, molecular moiety comprising suchas, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein.

In certain embodiments of Embodiment Set #19, the disruptedfurin-cleavage motif comprises an amino acid residue substitution in thefurin-cleavage motif relative to a wild-type Shiga toxin A Subunit. Incertain further embodiments, the substitution of the amino acid residuein the furin-cleavage motif is of an arginine residue with anon-positively charged, amino acid residue selected from the groupconsisting of: alanine, glycine, proline, serine, threonine, aspartate,asparagine, glutamate, glutamine, cysteine, isoleucine, leucine,methionine, valine, phenylalanine, tryptophan, and tyrosine. In certainembodiments, the substitution of the amino acid residue in thefurin-cleavage motif is of an arginine residue with a histidine.

In certain embodiments of Embodiment Set #19, the cell-targetingmolecule of the present invention is capable when introduced to cells ofexhibiting cytotoxicity that is greater than that of an eighteenthcell-targeting molecule having an amino-terminus and comprising thebinding region and the Shiga toxin effector polypeptide region which isnot positioned at or proximal to the amino-terminus of the eighteenthcell-targeting molecule. In certain further embodiments, thecell-targeting molecule of the present invention is capable ofexhibiting a cytotoxicity with better optimized, cytotoxic potency, suchas, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity ascompared to the eighteenth cell-targeting molecule. In certain furtherembodiments, the cytotoxicity of the cell-targeting molecule of thepresent invention to a population of target positive cells is 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater thanthe cytotoxicity of the eighteenth cell-targeting molecule to a secondpopulation of target positive cells as assayed by CD₅₀ values.

In certain embodiments of Embodiment Set #19, the cell-targetingmolecule is capable when introduced to a chordate of exhibitingimproved, in vivo tolerability compared to in vivo tolerability of anineteenth cell-targeting molecule consisting of the cell-targetingmolecule except for all of its Shiga toxin effector polypeptidecomponent(s) each comprise a wild-type Shiga toxin A1 fragment and/orwild-type Shiga toxin furin-cleavage site at the carboxy terminus of itsA1 fragment region.

In certain embodiments of Embodiment Set #19, the cell-targetingmolecule is de-immunized due to the furin-cleavage motif disruption. Incertain further embodiments, the cell-targeting molecule is capable ofexhibiting less relative antigenicity and/or relative immunogenicity ascompared to a reference cell-targeting molecule consisting of thecell-targeting molecule except for the furin-cleavage motif is wild-typeand/or all the Shiga toxin effector polypeptide components consist of awild-type Shiga toxin A1 fragment, such as, e.g., the nineteenthcell-targeting molecule.

For certain further embodiments of Embodiment Set #19, thecell-targeting molecule of the present invention is not cytotoxic and iscapable when introduced to cells of exhibiting a greater subcellularrouting efficiency from an extracellular space to a subcellularcompartment of an endoplasmic reticulum and/or cytosol as compared tothe cytotoxicity of a reference molecule, such as, e.g., the nineteenthcell-targeting molecule.

In certain embodiments of Embodiment Set #19, the cell-targetingmolecule of the present invention comprises two proteins selected fromany one of the polypeptides shown in any one of SEQ ID NOs: 252-255,259-271, 274-278 and 288-748, and which optionally comprises anamino-terminal methionine residue.

Embodiment Set #20—Multivalent Cell-Targeting Molecule Comprising aCarboxy-Terminal Endoplasmic Reticulum Retention/Retrieval Signal Motifand Shiga Toxin Effector Polypeptide at or Proximal to an Amino-Terminusof the Cell Targeting Molecule

The present invention provides multivalent cell-targeting molecules,each comprising (i) two or more binding regions, each capable ofspecifically binding an extracellular part of the same targetbiomolecule, (ii) a carboxy-terminal, endoplasmic reticulumretention/retrieval signal motif, and (iii) a Shiga toxin effectorpolypeptide; wherein the amino-terminus of the Shiga toxin effectorpolypeptide is at and/or proximal to an amino-terminus of a polypeptidecomponent of the cell-targeting molecule. In certain embodiments, thecell-targeting molecule of the present invention comprises a (i) bindingregion capable of specifically binding an extracellular targetbiomolecule, (ii) a carboxy-terminal, endoplasmic reticulumretention/retrieval signal motif of a member of the KDEL family, (iii) apolypeptide component, and (iv) a Shiga toxin effector polypeptide;wherein the amino-terminus of the Shiga toxin effector polypeptide is atand/or proximal to an amino-terminus of a polypeptide component of thecell-targeting molecule. In certain further embodiments, the bindingregion and Shiga toxin effector polypeptide are physically arranged ororiented within the cell-targeting molecule such that the binding regionis not located proximally to the amino-terminus of the Shiga toxineffector polypeptide. In certain further embodiments, the binding regionis located within the cell-targeting molecule more proximal to thecarboxy-terminus of the Shiga toxin effector polypeptide than to theamino-terminus of the Shiga toxin effector polypeptide. In certainfurther embodiments, the binding region is not located proximally to anamino-terminus of the cell-targeting molecule relative to the Shigatoxin effector polypeptide.

For certain further embodiments, the Shiga toxin effector polypeptide iscapable of exhibiting at least one Shiga toxin effector function, suchas, e.g., directing intracellular routing to the endoplasmic reticulumand/or cytosol of a cell in which the polypeptide is present, inhibitinga ribosome function, enzymatically inactivating a ribosome, causingcytostasis, and/or causing cytotoxicity. In certain further embodiments,the cell-targeting molecule of the present invention is capable of oneor more the following: entering a cell, inhibiting a ribosome function,causing cytostasis, and/or causing cell death.

In certain embodiments of Embodiment Set #20, the carboxy-terminalendoplasmic reticulum retention/retrieval signal motif is selected fromthe group consisting of: KDEL, HDEF, HDEL, RDEF, RDEL, WDEL, YDEL, HEEF,HEEL, KEEL, REEL, KAEL, KCEL, KFEL, KGEL, KHEL, KLEL, KNEL, KQEL, KREL,KSEL, KVEL, KWEL, KYEL, KEDL, KIEL, DKEL, FDEL, KDEF, KKEL, HADL, HAEL,HIEL, HNEL, HTEL, KTEL, HVEL, NDEL, QDEL, REDL, RNEL, RTDL, RTEL, SDEL,TDEL, SKEL, STEL, and EDEL.

In certain embodiments of Embodiment Set #20, the cell-targetingmolecule of the present invention is capable when introduced to cells ofexhibiting cytotoxicity that is greater than that of a twentiethcell-targeting molecule having an amino-terminus and comprising thebinding region and the Shiga toxin effector polypeptide region which isnot positioned at or proximal to the amino-terminus of the twentiethcell-targeting molecule and/or greater than that of a twenty-firstcell-targeting molecule consisting of the cell-targeting molecule exceptfor it does not comprise any carboxy-terminal, endoplasmic reticulumretention/retrieval signal motif of the KDEL family. In certain furtherembodiments, the twentieth cell-targeting molecule does not comprise anycarboxy-terminal, endoplasmic reticulum retention/retrieval signal motifof the KDEL family. In certain further embodiments, the cell-targetingmolecule of the present invention is capable of exhibiting acytotoxicity with better optimized, cytotoxic potency, such as, e.g.,4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity as compared to areference molecule, such as, e.g., the twentieth and/or twenty-firstcell-targeting molecules. In certain further embodiments, thecytotoxicity of the cell-targeting molecule of the present invention toa population of target positive cells is 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold or greater than the cytotoxicity of thetwentieth and/or twenty-first cell-targeting molecules to a secondpopulation of target positive cells as assayed by CD₅₀ values.

For certain further embodiments of Embodiment Set #20, thecell-targeting molecule of the present invention is not cytotoxic and iscapable when introduced to cells of exhibiting a greater subcellularrouting efficiency from an extracellular space to a subcellularcompartment of an endoplasmic reticulum and/or cytosol as compared tothe cytotoxicity of a reference molecule, such as, e.g., the twentiethand/or twenty-first cell-targeting molecules.

Further Embodiments of Embodiment Sets #1-#20

In certain embodiments of Embodiment Sets #1 to #20, the heterologous,CD8+ T-cell epitope-peptide cargo is fused, either directly orindirectly, to the Shiga toxin effector polypeptide and/or the bindingregion. In certain further embodiments, the cell-targeting moleculecomprises a single-chain polypeptide comprising the binding region, theShiga toxin effector polypeptide, and the heterologous, CD8+ T-cellepitope-peptide cargo.

In certain embodiments of Embodiment Sets #1 to #20, the binding regioncomprises two or more polypeptide chains and the heterologous, CD8+T-cell epitope-peptide cargo is fused either directly or indirectly, toa polypeptide comprising the Shiga toxin effector polypeptide and one ofthe two or more polypeptide chains of the binding region.

In certain embodiments of Embodiment Sets #1 to #20, the cell-targetingmolecule of the present invention comprises a heterologous, CD8+ T-cellepitope-peptide cargo which is positioned within the cell-targetingmolecule carboxy-terminal to the Shiga toxin effector polypeptide and/orbinding region. In certain further embodiments, the cell-targetingmolecule comprises two, three, four, five, or more heterologous, CD8+T-cell epitope-peptide cargos positioned within the cell-targetingmolecule carboxy-terminal to the Shiga toxin effector polypeptide and/orbinding region.

In certain embodiments of Embodiment Sets #1 to #20, the cell-targetingmolecule comprises a carboxy-terminal, heterologous, CD8+ T-cellepitope-peptide cargo.

For certain embodiments of Embodiment Sets #1 to #20, uponadministration of the cell-targeting molecule to a target cellphysically coupled with an extracellular target biomolecule of thebinding region, the cell-targeting molecule is capable of causingintercellular engagement of the target cell by a CD8+ immune cell.

For certain embodiments of Embodiment Sets #1 to #20, uponadministration of the cell-targeting molecule of the present inventionto a target cell physically coupled with an extracellular targetbiomolecule of the binding region, the cell-targeting molecule iscapable of causing intercellular engagement of the target cell by a CD8+immune cell. For certain further embodiments, upon administration of thecell-targeting molecule of the present invention to a target cellphysically coupled with an extracellular target biomolecule of thebinding region, the cell-targeting molecule is capable of causing deathof the target cell. For certain further embodiments, upon administrationof the cell-targeting molecule of the present invention to a firstpopulation of cells whose members are physically coupled toextracellular target biomolecules of the binding region, and a secondpopulation of cells whose members are not physically coupled to anyextracellular target biomolecule of the binding region, the cytotoxiceffect of the cell-targeting molecule to members of said firstpopulation of cells relative to members of said second population ofcells is at least 3-fold greater. In certain further embodiments, thecell-targeting molecule comprises or consists essentially of thepolypeptide of any one of SEQ ID NOs: 252-255, 259-278, and 288-748. Incertain embodiments, the Shiga toxin effector polypeptide comprises amutation relative to a naturally occurring A Subunit of a member of theShiga toxin family which changes the enzymatic activity of the Shigatoxin effector polypeptide, the mutation selected from at least oneamino acid residue deletion, insertion, or substitution. In certainfurther embodiments, the mutation is selected from at least one aminoacid residue deletion, insertion, or substitution that reduces oreliminates cytotoxicity of the toxin effector polypeptide. In certainembodiments, the binding region comprises the heterologous, CD8+ T-cellepitope cargo, whether the CD8+ epitope-peptide is autogenous orheterologous with respect to the binding region.

In certain embodiments of Embodiment Sets #1 to #20, the Shiga toxineffector polypeptide is fused to a binding region, either directly orindirectly, such as, e.g., via a linker known to the skilled worker.

In certain embodiments of Embodiment Sets #1 to #20, the cell-targetingmolecule comprises a molecular moiety located carboxy-terminal to thecarboxy-terminus of the Shiga toxin A1 fragment region.

In certain embodiments of Embodiment Sets #1 to #20, the Shiga toxineffector polypeptide has a Shiga toxin A1 fragment derived region havinga carboxy terminus and further comprises a disrupted furin-cleavagemotif at the carboxy-terminus of the A1 fragment region.

For certain embodiments of Embodiment Sets #1 to #20, the cell-targetingmolecule of the present invention is capable of exhibiting (i) acatalytic activity level comparable to a wild-type Shiga toxin A1fragment or wild-type Shiga toxin effector polypeptide, (ii) a ribosomeinhibition activity with a half-maximal inhibitory concentration (IC₅₀)value of 10,000 picomolar or less, and/or (iii) a significant level ofShiga toxin catalytic activity.

For certain embodiments of Embodiment Sets #1 to #20, the cell-targetingmolecule of the present invention is capable when introduced to cells ofexhibiting a cytotoxicity with a half-maximal inhibitory concentration(CD₅₀) value of 300 nM or less and/or capable of exhibiting asignificant level of Shiga toxin cytotoxicity.

For certain embodiments of Embodiment Sets #1 to #20, the cell-targetingmolecule of the present invention exhibits low cytotoxic potency (i.e.is not capable when introduced to certain positive target cell types ofexhibiting a cytotoxicity greater than 1% cell death of a cellpopulation at a cell-targeting molecule concentration of 100 nM, 500 nM,100 nM, 75 nM, or 50 nM).

In certain embodiments of Embodiment Sets #1 to #20, the cell-targetingmolecule of the present invention, or a polypeptide component thereof,comprises a carboxy-terminal, endoplasmic reticulum retention/retrievalsignal motif of a member of the KDEL family. For certain furtherembodiments, the carboxy-terminal endoplasmic reticulumretention/retrieval signal motif is selected from the group consistingof: KDEL, HDEF, HDEL, RDEF, RDEL, WDEL, YDEL, HEEF, HEEL, KEEL, REEL,KAEL, KCEL, KFEL, KGEL, KHEL, KLEL, KNEL, KQEL, KREL, KSEL, KVEL, KWEL,KYEL, KEDL, KIEL, DKEL, FDEL, KDEF, KKEL, HADL, HAEL, HIEL, HNEL. HTEL,KTEL, HVEL, NDEL, QDEL, REDL. RNEL, RTDL, RTEL, SDEL, TDEL, SKEL. STEL,and EDEL. In certain further embodiments, the cell-targeting molecule ofthe present invention is capable when introduced to cells of exhibitingcytotoxicity that is greater than that of a reference molecule, such as,e.g., a twenty-second cell-targeting molecule consisting of thecell-targeting molecule except for it does not comprise anycarboxy-terminal, endoplasmic reticulum retention/retrieval signal motifof the KDEL family. In certain further embodiments, the cell-targetingmolecule of the present invention is capable of exhibiting acytotoxicity with better optimized, cytotoxic potency, such as, e.g.,4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity as compared to areference molecule, such as, e.g., the twenty-second cell-targetingmolecule. In certain further embodiments, the cytotoxicity of thecell-targeting molecule of the present invention to a population oftarget positive cells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold or greater than the cytotoxicity of the twenty-secondcell-targeting molecule to a second population of target positive cellsas assayed by CD₅₀ values.

In certain embodiments of Embodiment Sets #1 to #20, the Shiga toxineffector polypeptide further comprises at least one inserted orembedded, heterologous epitope.

In certain embodiments of Embodiment Sets #1 to #20, the Shiga toxineffector polypeptide further comprises at least one, two, or threedisrupted, endogenous, B-cell and/or CD4+ T-cell epitope regions. Incertain further embodiments, the Shiga toxin effector polypeptidecomprises a disruption of at least one, two, or three endogenous, B-celland/or T-cell epitopes and/or epitope regions. In certain furtherembodiments, the Shiga toxin effector polypeptide further comprises atleast one disrupted, endogenous, B-cell and/or CD4+ T-cell epitoperegion which does not overlap with at least one inserted or embedded,heterologous epitope.

In certain embodiments of Embodiment #1 to #20, the amino-terminus ofthe Shiga toxin effector polypeptide is at and/or proximal to anamino-terminus of a polypeptide component of the cell-targetingmolecule. In certain further embodiments, the binding region is notlocated proximally to the amino-terminus of the cell-targeting moleculerelative to the Shiga toxin effector polypeptide. In certain furtherembodiments, the binding region and Shiga toxin effector polypeptide arephysically arranged or oriented within the cell-targeting molecule suchthat the binding region is not located proximally to the amino-terminusof the Shiga toxin effector polypeptide. In certain further embodiments,the binding region is located within the cell-targeting molecule moreproximal to the carboxy-terminus of the Shiga toxin effector polypeptidethan to the amino-terminus of the Shiga toxin effector polypeptide. Forcertain further embodiments, the cell-targeting molecule of the presentinvention is not cytotoxic and is capable when introduced to cells ofexhibiting a greater subcellular routing efficiency from anextracellular space to a subcellular compartment of an endoplasmicreticulum and/or cytosol as compared to the cytotoxicity of a referencemolecule, such as, e.g., a twenty-third cell-targeting molecule havingan amino-terminus and comprising the binding region and the Shiga toxineffector polypeptide which is not positioned at or proximal to theamino-terminus of the third cell-targeting molecule. For certain furtherembodiments, the cell-targeting molecule of the present inventionexhibits cytotoxicity with better optimized, cytotoxic potency, such as,e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity ascompared to the cytotoxicity of the twenty-third cell-targetingmolecule. For certain further embodiments, the cytotoxicity of thecell-targeting molecule of the present invention to a population oftarget positive cells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold or greater than the cytotoxicity of the twenty-thirdcell-targeting molecule to a second population of target positive cellsas assayed by CD₅₀ values. In certain further embodiments, thetwenty-third cell-targeting molecule does not comprise anycarboxy-terminal, endoplasmic reticulum retention/retrieval signal motifof the KDEL family.

In certain embodiments of Embodiment Sets #1 to #20, the Shiga toxineffector polypeptide further comprises a disruption in the B-cell and/orT-cell epitope region selected from the group of natively positionedShiga toxin A Subunit regions consisting of: 1-15 of any one of SEQ IDNOs: 1-2 and 4-6; 3-14 of any one of SEQ ID NOs: 3 and 7-18; 26-37 ofany one of SEQ ID NOs: 3 and 7-18; 27-37 of any one of SEQ ID NOs: 1-2and 4-6; 39-48 of any one of SEQ ID NOs: 1-2 and 4-6; 42-48 of any oneof SEQ ID NOs: 3 and 7-18; and 53-66 of any one of SEQ ID NOs: 1-18;94-115 of any one of SEQ ID NOs: 1-18; 141-153 of any one of SEQ ID NOs:1-2 and 4-6; 140-156 of any one of SEQ ID NOs: 3 and 7-18; 179-190 ofany one of SEQ ID NOs: 1-2 and 4-6; 179-191 of any one of SEQ ID NOs: 3and 7-18; 204 of SEQ ID NO:3; 205 of any one of SEQ ID NOs: 1-2 and 4-6;and 210-218 of any one of SEQ ID NOs: 3 and 7-18; 240-260 of any one ofSEQ ID NOs: 3 and 7-18; 243-257 of any one of SEQ ID NOs: 1-2 and 4-6;254-268 of any one of SEQ ID NOs: 1-2 and 4-6; 262-278 of any one of SEQID NOs: 3 and 7-18; 281-297 of any one of SEQ ID NOs: 3 and 7-18; and285-293 of any one of SEQ ID NOs: 1-2 and 4-6; or the equivalent regionin a Shiga toxin A Subunit or derivative thereof. In certain furtherembodiments, there is no disruption which is a carboxy-terminaltruncation of amino acid residues that overlap with part or all of atleast one disrupted, endogenous, B-cell and/or T-cell epitope and/orepitope region.

In certain embodiments of Embodiment Sets #1 to #20, the Shiga toxineffector polypeptide further comprises a mutation, relative to awild-type Shiga toxin A Subunit, in the B-cell immunogenic, amino acidresidue selected from the group of natively positioned Shiga toxin ASubunit amino acid residues: L49, D197, D198. R204, and R205.

In certain embodiments of Embodiment Sets #1 to #20, the embedded orinserted, heterologous, T-cell epitope disrupts the endogenous, B-celland/or T-cell epitope region is selected from the group of nativelypositioned Shiga toxin A Subunit regions consisting of: (i) 1-15 of anyone of SEQ ID NOs: 1-2 and 4-6; 3-14 of any one of SEQ ID NOs: 3 and7-18; 26-37 of any one of SEQ ID NOs: 3 and 7-18; 27-37 of any one ofSEQ ID NOs: 1-2 and 4-6; 39-48 of any one of SEQ ID NOs: 1-2 and 4-6;42-48 of any one of SEQ ID NOs: 3 and 7-18; and 53-66 of any one of SEQID NOs: 1-18, or the equivalent region in a Shiga toxin A Subunit orderivative thereof; (ii) 94-115 of any one of SEQ ID NOs: 1-18; 141-153of any one of SEQ ID NOs: 1-2 and 4-6; 140-156 of any one of SEQ ID NOs:3 and 7-18; 179-190 of any one of SEQ ID NOs: 1-2 and 4-6; 179-191 ofany one of SEQ ID NOs: 3 and 7-18; 204 of SEQ ID NO:3; 205 of any one ofSEQ ID NOs: 1-2 and 4-6; and 210-218 of any one of SEQ ID NOs: 3 and7-18, or the equivalent region in a Shiga toxin A Subunit or derivativethereof: and (iii) 240-260 of any one of SEQ ID NOs: 3 and 7-18; 243-257of any one of SEQ ID NOs: 1-2 and 4-6; 254-268 of any one of SEQ ID NOs:1-2 and 4-6; 262-278 of any one of SEQ ID NOs: 3 and 7-18; 281-297 ofany one of SEQ ID NOs: 3 and 7-18; and 285-293 of any one of SEQ ID NOs:1-2 and 4-6, or the equivalent region in a Shiga toxin A Subunit orderivative thereof.

In certain embodiments of Embodiment Sets #1 to #20, the Shiga toxineffector polypeptide comprises a mutation, relative to a wild-type Shigatoxin A Subunit, in the B-cell and/or T-cell epitope region selectedfrom the group of natively positioned Shiga toxin A Subunit regionsconsisting of: (i) 1-15 of any one of SEQ ID NOs: 1-2 and 4-6; 3-14 ofany one of SEQ ID NOs: 3 and 7-18; 26-37 of any one of SEQ ID NOs: 3 and7-18; 27-37 of any one of SEQ ID NOs: 1-2 and 4-6; 39-48 of any one ofSEQ ID NOs: 1-2 and 4-6; 42-48 of any one of SEQ ID NOs: 3 and 7-18; and53-66 of any one of SEQ ID NOs: 1-18, or the equivalent region in aShiga toxin A Subunit or derivative thereof, wherein there is nodisruption which is an amino-terminal truncation of sequences thatoverlap with part or all of at least one disrupted epitope region; (ii)94-115 of any one of SEQ ID NOs: 1-18; 141-153 of any one of SEQ ID NOs:1-2 and 4-6; 140-156 of any one of SEQ ID NOs: 3 and 7-18; 179-190 ofany one of SEQ ID NOs: 1-2 and 4-6; 179-191 of any one of SEQ ID NOs: 3and 7-18; 204 of SEQ ID NO:3; 205 of any one of SEQ ID NOs: 1-2 and 4-6;and 210-218 of any one of SEQ ID NOs: 3 and 7-18, or the equivalentregion in a Shiga toxin A Subunit or derivative thereof, wherein thereis no disruption which is an amino-terminal truncation of sequences thatoverlap with part or all of at least one disrupted epitope region.

In certain embodiments of Embodiment Sets #1 to #20, the Shiga toxineffector polypeptide comprises a disruption of at least one endogenousepitope region selected from the group of natively positioned Shigatoxin A Subunits consisting of: (ii) 94-115 of any one of SEQ ID NOs:1-18; 141-153 of any one of SEQ ID NOs: 1-2 and 4-6; 140-156 of any oneof SEQ ID NOs: 3 and 7-18; 179-190 of any one of SEQ ID NOs: 1-2 and4-6; 179-191 of any one of SEQ ID NOs: 3 and 7-18; 204 of SEQ ID NO:3;205 of any one of SEQ ID NOs: 1-2 and 4-6; and 210-218 of any one of SEQID NOs: 3 and 7-18, or the equivalent region in a Shiga toxin A Subunitor derivative thereof.

In certain embodiments of Embodiment Sets #2 to #11, the Shiga toxineffector polypeptide does not comprise a heterologous, MHC classI-restricted, T-cell epitope. MHC class I-restricted, T-cell epitopesare known in the art or can be predicted by the skilled worker. The termheterologous refers to MHC class I-restricted, T-cell epitopes which arenot natively present in wild-type Shiga toxin A Subunits, such as, e.g.,the wild-type Shiga toxin A Subunit which is most closely related to theShiga toxin effector polypeptide of interest.

In certain embodiments of Embodiment Sets #1 to #20, the Shiga toxineffector polypeptide comprises disruptions of at least four, five, six,seven, eight, or more endogenous, B-cell and/or T-cell epitope regions.

In certain embodiments of Embodiment Sets #1 to #20, one or moredisruptions comprises an amino acid residue substitution relative to awild-type Shiga toxin A Subunit.

In certain embodiments of Embodiment Sets #1 to #20, one or moreendogenous, B-cell and/or T-cell epitope regions comprises a pluralityof amino acid residue substitutions relative to a wild-type Shiga toxinA Subunit.

In certain embodiments of Embodiment Sets #1 to #20, at least one, two,three, or four disruptions comprise a plurality of amino acid residuesubstitutions in the endogenous, B-cell and/or T-cell epitope regionrelative to a wild-type Shiga toxin A Subunit.

In certain embodiments of Embodiment Sets #1 to #20, at least onedisruption comprises at least one, two, three, four, five, six, seven,eight, or more amino acid residue substitutions relative to a wild-typeShiga toxin A Subunit, and optionally wherein at least one substitutionoccurs at the natively positioned Shiga toxin A Subunit amino acidresidue selected form the group consisting of: 1 of SEQ ID NO: 1 or SEQID NO:2; 4 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 6 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 8 of SEQ ID NO: 1, SEQ ID NO:2, or SEQID NO:3; 9 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 11 of SEQ IDNO: 1, SEQ ID NO:2, or SEQ ID NO:3; 12 of SEQ ID NO: 1, SEQ ID NO:2, orSEQ ID NO:3; 33 of SEQ ID NO: 1 or SEQ ID NO:2; 43 of SEQ ID NO: 1 orSEQ ID NO:2; 44 of SEQ ID NO: 1 or SEQ ID NO:2; 45 of SEQ ID NO: 1 orSEQ ID NO:2; 46 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 47 of SEQID NO: 1 or SEQ ID NO:2; 48 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ IDNO:3; 49 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 50 of SEQ ID NO:1 or SEQ ID NO:2; 51 of SEQ ID NO:1 or SEQ ID NO:2; 53 of SEQ ID NO:1 orSEQ ID NO:2; 54 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 55 of SEQID NO:1 or SEQ ID NO:2; 56 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3;57 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 58 of SEQ ID NO: 1, SEQID NO:2, or SEQ ID NO:3; 59 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;60 of SEQ ID NO:1 or SEQ ID NO:2; 61 of SEQ ID NO: 1 or SEQ ID NO:2; 62of SEQ ID NO: 1 or SEQ ID NO:2; 84 of SEQ ID NO: 1 or SEQ ID NO:2; 88 ofSEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 94 of SEQ ID NO: 11, SEQ IDNO:2, or SEQ ID NO:3; 96 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;104 of SEQ ID NO: 1 or SEQ ID NO:2; 105 of SEQ ID NO: 1 or SEQ ID NO:2;107 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 108 of SEQ ID NO:1 orSEQ ID NO:2; 109 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 110 of SEQID NO:1 or SEQ ID NO:2; 111 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ IDNO:3; 112 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 141 of SEQ IDNO: 1, SEQ ID NO:2, or SEQ ID NO:3; 147 of SEQ ID NO: 1, SEQ ID NO:2, orSEQ ID NO:3; 154 of SEQ ID NO: 1 or SEQ ID NO:2; 179 of SEQ ID NO: 1,SEQ ID NO:2, or SEQ ID NO:3; 180 of SEQ ID NO: 1 or SEQ ID NO:2; 181 ofSEQ ID NO: 1 or SEQ ID NO:2; 183 of SEQ ID NO:1, SEQ ID NO:2, or SEQ IDNO:3; 184 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 185 of SEQ ID NO:1 or SEQ ID NO:2; 186 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 187of SEQ ID NO:1 or SEQ ID NO:2; 188 of SEQ ID NO: 1 or SEQ ID NO:2; 189of SEQ ID NO: 1 or SEQ ID NO:2; 197 of SEQ ID NO:3; 198 of SEQ ID NO: 1or SEQ ID NO:2; 204 of SEQ ID NO:3; 205 of SEQ ID NO: 1 or SEQ ID NO:2;247 of SEQ ID NO:1 or SEQ ID NO:2; 247 of SEQ ID NO:3; 248 of SEQ IDNO:1 or SEQ ID NO:2; 250 of SEQ ID NO:3; 251 of SEQ ID NO:1 or SEQ IDNO:2; 264 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 265 of SEQ IDNO:1 or SEQ ID NO:2; and 286 of SEQ ID NO:1 or SEQ ID NO:2; or theequivalent amino acid residue in a Shiga toxin A Subunit or derivativethereof. In certain further embodiments, at least two disruptions eachcomprise at least one amino acid residue substitutions relative to awild-type Shiga toxin A Subunit selected form the group consisting of 1of SEQ ID NO: 1 or SEQ ID NO:2; 4 of SEQ ID NO: 1, SEQ ID NO:2, or SEQID NO:3; 8 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 9 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3; 11 of SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3; 33 of SEQ ID NO: 1 or SEQ ID NO:2; 43 of SEQ ID NO: 1 orSEQ ID NO:2; 45 of SEQ ID NO:1 or SEQ ID NO:2; 47 of SEQ ID NO: 1 or SEQID NO:2; 48 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 49 of SEQ IDNO:1 or SEQ ID NO:2; 53 of SEQ ID NO: 1 or SEQ ID NO:2; 55 of SEQ IDNO:1 or SEQ ID NO:2; 58 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 59of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 60 of SEQ ID NO: 1 or SEQID NO:2; 61 of SEQ ID NO: 1 or SEQ ID NO:2; 62 of SEQ ID NO: 1 or SEQ IDNO:2; 94 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 96 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3; 109 of SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3; 110 of SEQ ID NO: 1 or SEQ ID NO:2; 112 of SEQ ID NO: 1,SEQ ID NO:2, or SEQ ID NO:3; 147 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ IDNO:3; 179 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 180 of SEQ IDNO:1 or SEQ ID NO:2; 181 of SEQ ID NO: 1 or SEQ ID NO:2; 183 of SEQ IDNO: 1, SEQ ID SEQ ID NO:2, or SEQ ID NO:3; 184 of SEQ ID NO: 1, SEQ IDNO:2, or SEQ ID NO:3; 185 of SEQ ID NO: 1 or SEQ ID NO:2; 186 of SEQ IDNO: 1, SEQ ID NO:2, or SEQ ID NO:3; 187 of SEQ ID NO:1 or SEQ ID NO:2;188 of SEQ ID NO:1 or SEQ ID NO:2; 189 of SEQ ID NO: 1 or SEQ ID NO:2;204 of SEQ ID NO:3; 205 of SEQ ID NO: 1 or SEQ ID NO:2; 247 of SEQ IDNO: 1 or SEQ ID NO:2; 247 of SEQ ID NO:3; 250 of SEQ ID NO:3; 264 of SEQID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 265 of SEQ ID NO:1 or SEQ IDNO:2; and 286 of SEQ ID NO:1 or SEQ ID NO:2; or the equivalent aminoacid residue in a Shiga toxin A Subunit or derivative thereof.

In certain embodiments of Embodiment Sets #1 to #20, the Shiga toxineffector polypeptide comprises disruption of at least three, endogenous.B-cell and/or T-cell epitope regions selected from the group ofconsisting of: (i) 1-15 of any one of SEQ ID NOs: 1-2 and 4-6; 3-14 ofany one of SEQ ID NOs: 3 and 7-18; 26-37 of any one of SEQ ID NOs: 3 and7-18; 27-37 of any one of SEQ ID NOs: 1-2 and 4-6; 39-48 of any one ofSEQ ID NOs: 1-2 and 4-6; 42-48 of any one of SEQ ID NOs: 3 and 7-18; and53-66 of any one of SEQ ID NOs: 1-18, or the equivalent region in aShiga toxin A Subunit or derivative thereof, wherein there is nodisruption which is an amino-terminal truncation of amino acid residuesthat overlap with part or all of at least one disrupted, endogenous.B-cell and/or T-cell epitope region: (ii) 94-115 of any one of SEQ IDNOs: 1-18; 141-153 of any one of SEQ ID NOs: 1-2 and 4-6; 140-156 of anyone of SEQ ID NOs: 3 and 7-18; 179-190 of any one of SEQ ID NOs: 1-2 and4-6; 179-191 of any one of SEQ ID NOs: 3 and 7-18; 204 of SEQ ID NO:3;205 of any one of SEQ ID NOs: 1-2 and 4-6; and 210-218 of any one of SEQID NOs: 3 and 7-18, or the equivalent region in a Shiga toxin A Subunitor derivative thereof, wherein there is no disruption which is acarboxy-terminal truncation of amino acid residues that overlap withpart or all of at least one disrupted, endogenous, B-cell and/or T-cellepitope and/or epitope region.

In certain embodiments of Embodiment Sets #1 to #20, the Shiga toxineffector polypeptide comprises disruptions of at least two, endogenous.B-cell and/or T-cell epitope regions, wherein each disruption comprisesone or more amino acid residue substitutions, and wherein theendogenous, B-cell and/or T-cell epitope regions are selected from thegroup of natively positioned Shiga toxin A Subunit regions consistingof: 3-14 of any one of SEQ ID NOs: 3 and 7-18; 26-37 of any one of SEQID NOs: 3 and 7-18; 27-37 of any one of SEQ ID NOs: 1-2 and 4-6; 39-48of any one of SEQ ID NOs: 1-2 and 4-6; 42-48 of any one of SEQ ID NOs: 3and 7-18; 53-66 of any one of SEQ ID NOs: 1-18; or the equivalent regionin a Shiga toxin A Subunit or derivative thereof.

In certain embodiments of Embodiment Sets #1 to #20, the embedded orinserted, heterologous, T-cell epitope does not disrupt any endogenous,B-cell and/or CD4+ T-cell epitope region described herein.

In certain embodiments of Embodiment Sets #1 to #20, at least onedisruption comprises one or more amino acid residue substitutionsrelative to a wild-type Shiga toxin A Subunit is selected from the groupconsisting of: D to A, D to G, D to V, D to L, D to I, D to F, D to S, Dto Q, D to M. D to R, E to A E to G, E to V, E to L, E to I, E to F, Eto S, E to Q, E to N, E to D, E to M, E to R, F to A, F to G, F to V, Fto L, F to I, G to A G to P, H to A H to G, H to V, H to L, H to I, H toF, H to M, I to A, I to V, I to G, I to C, K to A, K to G, K to V, K toL, K to I, K to M, K to H, L to A, L to V, L to G, L to C, N to A, N toG, N to V N to L, N to I, N to F, P to A, P to G, P to F, R to A, R toG, R to V, R to L, R to I, R to F, R to M, R to Q, R to S, R to K, R toH, S to A, S to G, S to V, S to L, S to I, S to F, S to M, T to A, T toG, T to V, T to L, T to I, T to F, T to M, T to S, V to A, V to G, Y toA, Y to G, Y to V, Y to L, Y to 1, Y to F, Y to M, and Y to T. Incertain further embodiments, the one or more amino acid residuesubstitutions relative to a wild-type Shiga toxin A Subunit is selectedfrom the group consisting of: D to A, D to G, D to V, D to L, D to I, Dto F, D to S, D to Q, E to A, E to G, E to V, E to L, E to I, E to F, Eto S, E to Q, E to N, E to D, E to M, E to R, G to A, H to A, H to G, Hto V, H to L, H to I, H to F, H to M, K to A, K to G, K to V, K to L, Kto I, K to M, K to H, L to A, L to G, N to A, N to G, N to V, N to L, Nto I, N to F, P to A, P to G, P to F, R to A, R to G, R to V, R to L, Rto I, R to F, R to M, R to Q R to S, R to K, R to H, S to A, S to G, Sto V, S to L, S to I, S to F, S to M, T to A, T to G, T to V, T to L, Tto I, T to F, T to M, T to S, Y to A, Y to G, Y to V, Y to L, Y to I, Yto F, and Y to M.

In certain embodiments of Embodiment Sets #1 to #20, at least one of thedisruption(s) comprises one or more amino acid residue substitutionsrelative to a wild-type Shiga toxin A Subunit selected from the groupconsisting of: K1 to A, G, V, L, I, F, M and H; T4 to A, G, V, L, I, F,M, and S; D6 to A, G, V, L, I, F, S, Q and R; S8 to A, G, V, I, L, F,and M; T9 to A, G, V, I, L, F M, and S; S9 to A, G, V, L, I, F, and M;K11 to A, G, V, L, I, F, M and H; T12 to A, G, V, I, L, F, M, S, and K;S12 to A, G, V, I, L, F, and M; S33 to A, G, V, L, I, F, M, and C; S43to A, G, V, L, I, F, and M; G44 to A or L; S45 to A, G, V, L, I, F, andM; T45 to A, G, V, L, I, F, and M; G46 to A and PD47 to A, G, V, L, I,F, S, M, and Q; N48 to A, G, V, L, M and F; L49 to A, V, C, and G; Y49to A, G, V, L, I, F, M, and T; F50 to A, G, V, L, I, and T; A51; D53 toA, G, V, L, I, F, S, and Q; V54 to A, G, I, and L; R55 to A, G, V, L, 1,F, M, Q, S, K, and H; G56 to A and P; 157 to A, G, V, and M; L57 to A,V, C, G, M and F; D58 to A, G, V, L, I, F, S, and Q; P59 to A, G, and F;E60 to A, G, V, L, I, F, S, Q, N, D, M, T, and R; E61 to A, G, V, L, I,F, S, Q, N, D, M, and R; G62 to A; R84 to A, G, V, L, I, F, M, Q, S, K,and H; V88 to A and G; 188 to A, V, C, and G; D94 to A, G, V, L, I, F,S, and Q; S96 to A, G, V, I, L, F, and M; T104 to A, G, V, L, I, F, M;and N; A105 to L; T107 to A, G, V, L, I, F, M, and P; S107 to A, G, V,L, I, F, M, and P; L108 to A, V, C, and G S109 to A, G, V, I, L, F, andM; T109 to A, G, V, I, L, F, M, and S; G10 to A; S112 to A, G, V, L, I,F, and M; D111 to A, G, V, L, I, F, S, Q, and T; S112 to A, G, V, L, I,F, and M; D141 to A, G, V, L, 1, F, S, and Q; G147 to A; V154 to A andG, R179 to A, G, V, L, I, F, M, Q, S, K, and H; T180 to A, G, V, L, I,F, M, and S; T181 to A, G, V, L, I, F, M, and S; D183 to A, G, V, L, I,F, S, and Q, D184 to A, G, V, L1, F, S, and Q; L185 to A, G, V and C;S186 to A, G, V, I, L, F, and M; G187 to A; R188 to A, G, V, L, I, F, M,Q, S, K, and H; S189 to A, G, V, I, L, F, and M; D197 to A, G, V, L, 1,F, S, and Q; D198 to A, G, V, L, 1, F, S, and Q; R204 to A, G, V, L, 1,F, M, Q, S, K, and H; R205 to A, G, V, L, 1, F, M, Q, S, K and H; S247to A, G, V, I, L, F, and M; Y247 to A, G, V, L, 1, F, and M; R248 to A,G, V, L, I, F, M, Q, S, K, and H; R250 to A, G, V, L, 1, F, M, Q, S, K,and H, R251 to A, G, V, L, I, F, M, Q, S, K, and H; D264 to A, G, V, L,1, F, S, and Q; G264 to A; and T286 to A, G, V, L, 1, F, M, and S,

For certain embodiments of Embodiment Sets #1 to #20, the cell-targetingmolecule of the present invention is capable when introduced to achordate of exhibiting improved in vivo tolerability and/or stabilitycompared to a reference molecule, such as, e.g., a twenty-fourthcell-targeting molecule consisting of the cell-targeting molecule exceptfor all of its Shiga toxin effector polypeptide component(s) eachcomprise a wild-type Shiga toxin A1 fragment and/or wild-type Shigatoxin furin-cleavage site at the carboxy terminus of its A1 fragmentregion. In certain further embodiments, the Shiga toxin effectorpolypeptide is not cytotoxic and the molecular moiety is cytotoxic.

In certain embodiments of Embodiment Sets #1 to #20, the binding regionand Shiga toxin effector polypeptide are linked together, eitherdirectly or indirectly.

In certain embodiments of Embodiment Sets #1 to #20, the binding regioncomprises at least one peptide and/or polypeptide. In certain furtherembodiments, the binding region is or comprises an immunoglobulin-typebinding region. In certain further embodiments, the binding regioncomprising a polypeptide selected from the group consisting of anautonomous V_(H) domain, single-domain antibody fragment (sdAb),nanobody, heavy chain-antibody domain derived from a camelid (V_(H)H orV_(H) domain fragment), heavy-chain antibody domain derived from acartilaginous fish (V_(H)H or V_(H) domain fragment), immunoglobulin newantigen receptor (IgNAR), V_(NAR) fragment, single-chain variablefragment (scFv), antibody variable fragment (Fv), complementarydetermining region 3 fragment (CDR3), constrained FR3-CDR3-FR4polypeptide (FR3-CDR3-FR4), Fd fragment, small modularimmunopharmaceutical (SMIP) domain, antigen-binding fragment (Fab),Armadillo repeat polypeptide (ArmRP), fibronectin-derived 10^(th)fibronectin type III domain (10Fn3), tenascin type III domain (TNfn3),ankyrin repeat motif domain, low-density-lipoprotein-receptor-derivedA-domain (LDLR-A), lipocalin (anticalin), Kunitz domain,Protein-A-derived Z domain, gamma-B crystallin-derived domain,ubiquitin-derived domain. Sac7d-derived polypeptide (affitin),Fyn-derived SH2 domain, miniprotein. C-type lectin-like domain scaffold,engineered antibody mimic, and any genetically manipulated counterpartsof any of the foregoing which retain binding functionality.

For certain embodiments of Embodiment Sets #1 to #20, the cell-targetingmolecule of the present invention is capable of exhibiting (i) acatalytic activity level comparable to a wild-type Shiga toxin A1fragment or wild-type Shiga toxin effector polypeptide, (ii) a ribosomeinhibition activity with a half-maximal inhibitory concentration (IC₅₀)value of 10,000 picomolar or less, and/or (iii) a significant level ofShiga toxin catalytic activity.

For certain embodiments of Embodiment Sets #1 to #20, the cell-targetingmolecule of the present invention and/or its Shiga toxin effectorpolypeptide is capable of exhibiting subcellular routing efficiencycomparable to a reference cell-targeting molecule comprising a wild-typeShiga toxin A1 fragment or wild-type Shiga toxin effector polypeptideand/or capable of exhibiting a significant level of intracellularrouting activity to the endoplasmic reticulum and/or cytosol from anendosomal starting location of a cell.

For certain embodiments of Embodiment Sets #1 to #20, wherebyadministration of the cell-targeting molecule of the present inventionto a cell physically coupled with the extracellular target biomoleculeof the cell-targeting molecule's binding region, the cell-targetingmolecule is capable of causing death of the cell. In certain furtherembodiments, administration of the cell-targeting molecule of theinvention to two different populations of cell types which differ withrespect to the presence or level of the extracellular targetbiomolecule, the cell-targeting molecule is capable of causing celldeath to the cell-types physically coupled with an extracellular targetbiomolecule of the cytotoxic cell-targeting molecule's binding region ata CD₅₀ at least three times or less than the CD₅₀ to cell types whichare not physically coupled with an extracellular target biomolecule ofthe cell-targeting molecule's binding region. For certain embodiments,whereby administration of the cell-targeting molecule of the presentinvention to a first populations of cells whose members are physicallycoupled to extracellular target biomolecules of the cell-targetingmolecule's binding region, and a second population of cells whosemembers are not physically coupled to any extracellular targetbiomolecule of the binding region, the cytotoxic effect of thecell-targeting molecule to members of said first population of cellsrelative to members of said second population of cells is at least3-fold greater. For certain embodiments, whereby administration of thecell-targeting molecule of the present invention to a first populationsof cells whose members are physically coupled to a significant amount ofthe extracellular target biomolecule of the cell-targeting molecule'sbinding region, and a second population of cells whose members are notphysically coupled to a significant amount of any extracellular targetbiomolecule of the binding region, the cytotoxic effect of thecell-targeting molecule to members of said first population of cellsrelative to members of said second population of cells is at least3-fold greater. For certain embodiments, whereby administration of thecell-targeting molecule of the present invention to a first populationof target biomolecule positive cells, and a second population of cellswhose members do not express a significant amount of a targetbiomolecule of the cell-targeting molecule's binding region at acellular surface, the cytotoxic effect of the cell-targeting molecule tomembers of the first population of cells relative to members of thesecond population of cells is at least 3-fold greater.

For certain embodiments of Embodiment Sets #1 to #20, the cell-targetingmolecule of the present invention is capable when introduced to cells ofexhibiting a cytotoxicity with a half-maximal inhibitory concentration(CD5) value of 300 nM or less and/or capable of exhibiting a significantlevel of Shiga toxin cytotoxicity.

For certain embodiments of Embodiment Sets #1 to #20, the cell-targetingmolecule of the present invention is capable of delivering an embeddedor inserted, heterologous, CD8+ T-cell epitope to a MHC class Ipresentation pathway of a cell for cell-surface presentation of theepitope bound by a MHC class I molecule.

In certain embodiments of Embodiment Sets #1 to #20, the cell-targetingmolecule comprises a molecular moiety associated with thecarboxy-terminus of the Shiga toxin effector polypeptide. In certainembodiments, the molecular moiety comprises or consists of the bindingregion. In certain embodiments, the molecular moiety comprises at leastone amino acid and the Shiga toxin effector polypeptide is linked to atleast one amino acid residue of the molecular moiety. In certain furtherembodiments, the molecular moiety and the Shiga toxin effectorpolypeptide are fused forming a continuous polypeptide.

In certain embodiments of Embodiment Sets #1 to #20, the cell-targetingmolecule further comprises a cytotoxic molecular moiety associated withthe carboxy-terminus of the Shiga toxin effector polypeptide. Forcertain embodiments, the cytotoxic molecular moiety is a cytotoxicagent, such as, e.g., a small molecule chemotherapeutic agent,anti-neoplastic agent, cytotoxic antibiotic, alkylating agent,antimetabolite, topoisomerase inhibitor, and/or tubulin inhibitor knownto the skilled worker and/or described herein. For certain furtherembodiments, the cytotoxic molecular moiety is cytotoxic atconcentrations of less than 10,000, 5,000, 1,000, 500, or 200 pM.

In certain embodiments of Embodiment Sets #1 to #20, the binding regionis capable of binding to an extracellular target biomolecule selectedfrom the group consisting of CD20, CD22, CD40, CD74, CD79, CD25, CD30,HER2/neu/ErbB2, EGFR, EpCAM, EphB2, prostate-specific membrane antigen,Cripto, CDCP1, endoglin, fibroblast activated protein, Lewis-Y, CD19,CD21, CS1/SLAMF7, CD33, CD52, CD133, CEA, gpA33, mucin, TAG-72,tyrosine-protein kinase transmembrane receptor (ROR1 or NTRKR1),carbonic anhydrase IX, folate binding protein, ganglioside GD2,ganglioside GD3, ganglioside GM2, ganglioside Lewis-Y2, VEGFR. Alpha Vbeta3, Alpha5beta1, ErbB1/EGFR. Erb3, c-MET, IGF1R, EphA3, TRAIL-R1,TRAIL-R2. RANK, FAP, tenascin, CD64, mesothelin, BRCA1, MART-1/MelanA,gp100, tyrosinase. TRP-1, TRP-2, MAGE-1, MAGE-3, GAGE-1/2, BAGE, RAGE,NY-ESO-1, CDK-4, beta-catenin, MUM-1, caspase-8, KIAA0205, HPVE6,SART-1, PRAME, carcinoembryonic antigen, prostate specific antigen,prostate stem cell antigen, human aspartyl (asparaginyl)beta-hydroxylase, EphA2, HER3/ErbB-3, MUC1, MART-1/MelanA, gp100,tyrosinase associated antigen. HPV-E7, Epstein-Barr virus antigen,Bcr-Abl, alpha-fetoprotein antigen, 17-A1, bladder tumor antigen, SAIL,CD38, CD15, CD23, CD45 (protein tyrosine phosphatase receptor type C),CD53, CD88, CD129, CD183, CD191, CD193, CD244, CD294, CD305, C3AR,FceRIa, galectin-9, IL-1R (interleukin-1 receptor), mrp-14, NKG2Dligand, programmed death-ligand 1 (PD-L1). Siglec-8, Siglec-10, CD49d,CD13, CD44, CD54, CD63, CD69, CD123, TLR4, FceRIa, IgE, CD107a, CD203c,CD14, CD68, CD80, CD86, CD105, CD115, F4/80, ILT-3, galectin-3, CD11a-c,GITRL, MHC class I molecule, MHC class II molecule (optionally complexedwith a peptide), CD284 (TLR4), CD107-Mac3, CD195 (CCR5), HLA-DR,CD16/32, CD282 (TLR2), CD11c, and any immunogenic fragment of any of theforegoing.

In certain embodiments of Embodiment Sets #1 to #20, the binding regionis linked, either directly or indirectly, to the Shiga toxin effectorpolypeptide by at least one covalent bond which is not a disulfide bond.In certain further embodiments, the binding region is fused, eitherdirectly or indirectly, to the carboxy-terminus of the Shiga toxineffector polypeptide to form a single, continuous polypeptide. Incertain further embodiments, the binding region is animmunoglobulin-type binding region.

In certain embodiments of Embodiment Sets #1 to #20, the disruptedfurin-cleavage motif comprises one or more mutations in the minimal,furin-cleavage site relative to a wild-type Shiga toxin A Subunit. Incertain embodiments, the disrupted furin-cleavage motif is not anamino-terminal truncation of sequences that overlap with part or all ofat least one amino acid residue of the minimal furin-cleavage site.

In certain embodiments, the mutation in the minimal, furin-cleavage siteis an amino acid deletion, insertion, and/or substitution of at leastone amino acid residue in the R/Y-x-x-R furin cleavage motif. In certainfurther embodiments, the disrupted furin-cleavage motif comprises atleast one mutation relative to a wild-type Shiga toxin A Subunit, themutation altering at least one amino acid residue in the region nativelypositioned at 248-251 of the A Subunit of Shiga toxin (SEQ ID NOs: 1-2and 4-6), or at 247-250 of the A Subunit of Shiga-like toxin 2 (SEQ IDNOs: 3 and 7-18), or the equivalent amino acid sequence position in anyShiga toxin A Subunit.

In certain further embodiments, the mutation is an amino acid residuesubstitution of an arginine residue with a non-positively charged, aminoacid residue.

In certain embodiments of Embodiment Sets #1 to #20, the cell-targetingmolecule of the present invention is capable when introduced to cells ofexhibiting cytotoxicity comparable to a cytotoxicity of a referencemolecule, such as, e.g., a twenty-fifth cell-targeting moleculeconsisting of the cell-targeting molecule except for all of its Shigatoxin effector polypeptide component(s) each comprise a wild-type Shigatoxin A1 fragment.

In certain embodiments of Embodiment Sets #1 to #20, one or more bindingregion(s) comprises the peptide or polypeptide shown in any one of SEQID NOs: 39-245.

In certain embodiments of Embodiment Sets #11 to #20, two or morebinding regions comprise the peptide or polypeptide shown in any one ofSEQ ID NOs: 39-245. In certain further embodiments of Embodiment Sets#11 to #20, two or more binding regions each comprise the same peptideor polypeptide shown in any one of SEQ ID NOs: 39-245.

Certain embodiments of the cell-targeting molecule of the presentinvention comprises any one of SEQ ID NOs: 19-255, 259-278, and 288-748.

Certain embodiments of the cell-targeting molecule of the presentinvention comprise or consist essentially of the polypeptide representedby the amino acid sequence shown in any one of SEQ ID NOs: 252-255,259-278, and 288-748.

In certain embodiments of Embodiment Sets #1 to #20, at least onebinding region sterically covers the carboxy-terminus of the A1 fragmentregion.

In certain embodiments of Embodiment Sets #1 to #20, the molecularmoiety sterically covers the carboxy-terminus of the A1 fragment region.In certain further embodiments, the molecular moiety comprises thebinding region.

In certain embodiments of Embodiment Sets #1 to #20, the cell-targetingmolecule of the present invention comprises a binding region and/ormolecular moiety located carboxy-terminal to the carboxy-terminus of theShiga toxin A1 fragment region. In certain further embodiments, the massof the binding region and/or molecular moiety is at least 4.5 kDa, 6,kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa,100 kDa, or greater.

In certain embodiments of Embodiment Sets #1 to #20, the cell-targetingmolecule comprises a binding region with a mass of at least 4.5 kDa, 6,kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50kDa, 100 kDa, or greater, as long as the cell-targeting molecule retainsthe appropriate level of the Shiga toxin biological activity notedherein (e.g., cytotoxicity and/or intracellular routing).

In certain embodiments of Embodiment Sets #1 to #20, the binding regionis comprised within a relatively large, molecular moiety comprising suchas, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein.

For certain embodiments of Embodiment Sets #1 to #20, the cell-targetingmolecule of the present invention exhibits low cytotoxic potency (i.e.is not capable when introduced to certain positive target cell types ofexhibiting a cytotoxicity greater than 1% cell death of a cellpopulation at a cell-targeting molecule concentration of 100 nM, 500 nM,100 nM, 75 nM, or 50 nM) and is capable when introduced to cells ofexhibiting a greater subcellular routing efficiency from anextracellular space to a subcellular compartment of an endoplasmicreticulum and/or cytosol as compared to the cytotoxicity of a referencemolecule, such as, e.g., a twenty-sixth cell-targeting molecule havingan amino-terminus and comprising the binding region and the Shiga toxineffector polypeptide which is not positioned at or proximal to theamino-terminus of the third cell-targeting molecule. In certain furtherembodiments, the twenty-sixth cell-targeting molecule does not compriseany carboxy-terminal, endoplasmic reticulum retention/retrieval signalmotif of the KDEL family.

In certain embodiments of Embodiment Sets #1 to #20, In certain furtherembodiments, the molecular moiety comprises a peptide and/or polypeptidederived from the Shiga toxin A2 fragment of a naturally occurring Shigatoxin.

The embodiments of the present invention are not intended to cover anynaturally-occurring Shiga holotoxin or Shiga toxin A Subunit. In certainembodiments of Embodiment Sets #1 to #20, the cell-targeting molecule ofthe present invention does not comprise a naturally occurring Shigatoxin B Subunit. In certain further embodiments, the cell-targetingmolecule of the invention does not comprise any polypeptide comprisingor consisting essentially of a functional binding domain of a nativeShiga toxin B subunit. Rather, in certain embodiments of thecell-targeting molecules of the invention, the Shiga toxin A Subunitderived regions are functionally associated with heterologous bindingregions to effectuate cell-targeting.

In certain embodiments of Embodiment Sets #1 to #20, the cell-targetingmolecule of the present invention is limited by the proviso that theheterologous, CD8+ T-cell epitope-peptide cargo does not comprise orconsist of the polypeptide shown in SEQ ID NO:25 for all variationsdescribed above of Embodiment Sets #1 to #20. In certain embodiments ofEmbodiment Sets #1 to #20, the cell-targeting molecule of the presentinvention is limited by the proviso that the cell-targeting molecule ofthe present invention does not comprise the Shiga toxin effectorpolypeptide comprising the CD8+ T-cell epitope-peptide GILGFVFTL (SEQ IDNO:25) embedded at native position 53 in SLT-1A (SEQ ID NO: 1) for allvariations described above of Embodiment Sets #1 to #20. In certainembodiments of Embodiment Sets #1 to #20, the cell-targeting molecule ofthe present invention is limited by the proviso that the cell-targetingmolecule of the present invention does not comprise the polypeptideshown in SEQ ID NO:25 for all variations described above of EmbodimentSets #1 to #20. In certain embodiments of Embodiment Sets #1 to #20, thecell-targeting molecule of the present invention is limited by theproviso that the cell-targeting molecule of the present invention doesnot comprise any Shiga toxin effector polypeptide comprising anyembedded or inserted, CD8+ T-cell epitope for all variations describedabove of Embodiment Sets #1 to #20.

In certain embodiments of Embodiment Sets #1 to #20, the cell-targetingmolecule of the present invention is limited by the proviso that thecell-targeting molecule of the present invention does not comprise thelinker shown in SEQ ID NO:247 wherein the linker is fused, eitherdirectly or indirectly, between a binding region and a Shiga toxineffector polypeptide and wherein the binding region is positionedamino-terminal to the Shiga toxin effector polypeptide for allvariations described above of Embodiment Sets #1 to #20. In certainembodiments of Embodiment Sets #1 to #20, the cell-targeting molecule ofthe present invention is limited by the proviso that the cell-targetingmolecule of the present invention does not comprise the linker shown inSEQ ID NO:247 wherein the linker is fused between a binding region and aShiga toxin effector polypeptide for all variations described above ofEmbodiment Sets #1 to #20.

In certain embodiments of Embodiment Sets #1 to #20, the cell-targetingmolecule of the present invention is limited by the proviso that thecell-targeting molecule of the present invention does not comprise orconsist essentially of the polypeptide shown in any one of SEQ ID NOs:259-278 and 287 for all variations described above of Embodiment Sets #1to #20.

In certain embodiments of Embodiment Sets #1 to #20, the cell-targetingmolecule of the present invention does not comprise any heterologous,CD8+ T-cell epitope-peptide cargo fused between a binding region and aShiga toxin effector polypeptide wherein the binding region ispositioned amino-terminal to the Shiga toxin effector. In certainembodiments, the cell-targeting molecule of the present invention doesnot comprise any heterologous, CD8+ T-cell epitope-peptide cargo fusedbetween a binding region and a Shiga toxin effector polypeptide.

For certain embodiments of Embodiment Sets #1 to #20, the target cell isnot a professional antigen presenting cell, such as a dendritic celltype. For certain embodiments of the cell-targeting molecule of thepresent invention, the extracellular target biomolecule of the bindingregion is not expressed by a professional antigen presenting cell. Forcertain embodiments of the cell-targeting molecule of the presentinvention, the extracellular target biomolecule of the binding region isnot physically associated in significant quantities with a professionalantigen presenting cell. For certain embodiments of the cell-targetingmolecule of the present invention, the extracellular target biomoleculeof the binding region is not physically associated with a professionalantigen presenting cell. For certain embodiments of the cell-targetingmolecules of the present invention, the target biomolecule of thebinding region is not expressed in significant amounts on the cellularsurface of any professional antigen presenting cell within the chordatesubject to be treated.

In certain embodiments of Embodiment Sets #1 to #20, the heterologous,CD8+ T-cell epitope-peptide cargo is not directly associated with anyamino acid residue of the Shiga toxin A1 fragment derived region of theShiga toxin effector polypeptide. In certain embodiments of thecell-targeting molecule of the present invention, the heterologous, CD8+T-cell epitope-peptide cargo is not directly associated with anyinternal amino acid residue of the Shiga toxin effector polypeptide,meaning either the amino- or carboxy-terminal amino acid residue of theShiga toxin effector polypeptide may be directly linked to aheterologous, CD8+50 T-cell epitope-peptide cargo.

In certain embodiments of Embodiment Sets #1 to #20, the binding regiondoes not comprise a fragment of human CD4 corresponding to amino acidresidues 19-183. In certain further embodiments, the binding region doesnot comprise a fragment of human CD4, a type-I transmembraneglycoprotein. In certain further embodiments, the binding region doesnot comprise a fragment of a human, immune cell surface co-receptor.

In certain embodiments of Embodiment Sets #1 to #20, the cell-targetingmolecule of the present invention does not comprise a carboxy-terminal,binding region comprising a fragment of an immune cell surface receptor.

In certain embodiments of Embodiment Sets #1 to #20, the Shiga toxineffector polypeptide comprises at least two, embedded or inserted,heterologous epitopes.

In certain embodiments of Embodiment Sets #1 to #20, the Shiga toxineffector polypeptide does not comprise the set of amino acid residuesubstitutions relative to a wild-type Shiga toxin A Subunit selectedfrom the following sets; (1) R248H and R251H; (2) R248G and R251G; (3)A246G, S247A, A253G, and S254A; and (4) A246G, S247A, R248G, R251G,A253G, and S254A.

In certain embodiments of Embodiment Sets #1 to #20, the Shiga toxineffector polypeptide does not comprise a deletion of the region nativelypositioned at 247-252 in a wild-type Shiga toxin A Subunit. In certainembodiments of Embodiment Sets #1 to #20, the Shiga toxin effectorpolypeptide does not comprise deletions of the regions nativelypositioned at 245-247 and 253-255 in a wild-type Shiga toxin A Subunit.

In certain embodiments of Embodiment Sets #1 to #20, the Shiga toxineffector polypeptide comprises one or more mutations relative to anaturally occurring A Subunit of a member of the Shiga toxin familywhich changes an enzymatic activity of the Shiga toxin effectorpolypeptide, the mutation selected from at least one amino acid residuedeletion, insertion, or substitution. In certain further embodiments,the mutation relative to the naturally occurring A Subunit reduces ofeliminates a cytotoxic activity of the Shiga toxin effector polypeptidebut the Shiga toxin effector polypeptide retains at least one otherShiga toxin effector function, such as, e.g., promoting cellularinternalization and/or directing intracellular routing to a certainsubcellular compartment(s). In certain further embodiments, the mutationrelative to the naturally occurring A Subunit is selected from at leastone amino acid residue substitution, such as, e.g., A231E, N75A, Y77S,Y114S, E167D, R170A, R176K, W202A, and/or W203A in SEQ ID NO: 1-18.

For certain embodiments of Embodiment Sets #1 to #20, the Shiga toxineffector polypeptide is capable of (i) routing to a subcellularcompartment of a cell in which the Shiga toxin effector polypeptide ispresent selected from the following: cytosol, endoplasmic reticulum, andlysosome; (ii) intracellular delivery of the epitope-cargo from an earlyendosomal compartment to a proteasome of a cell in which the Shiga toxineffector polypeptide is present; and/or (iii) intracellular delivery ofthe epitope to a MHC class I molecule from an early endosomalcompartment of a cell in which the Shiga toxin effector polypeptide ispresent. In certain further embodiments, the Shiga toxin effectorpolypeptide is capable of intracellular delivery of the CD8+ T-cellepitope for presentation by a MHC class I molecule on the surface of acell in which the Shiga toxin effector polypeptide is present.

In certain embodiments, the molecule of the present invention does notcomprise, at a position carboxy-terminal of the Shiga toxin effectorpolypeptide and/or the carboxy-terminus of the Shiga toxin A1 fragmentregion, any additional exogenous material representing an antigen and/orheterologous, CD8+, T-cell epitope-peptide cargo.

In certain embodiments of Embodiment Sets #1 to #20, the binding regiondoes not comprise a ligand. In certain embodiments of Embodiment Sets #1to #20, the binding region does not comprise a chemokine or aTNF-related apoptosis-inducing ligand (TRAIL) nor a receptor bindingfragment thereof. In certain embodiments of Embodiment Sets #1 to #20,the binding region does not comprise a human chemokine or human TRAILnor a receptor binding fragment thereof. In embodiments of EmbodimentSets #1 to #20, the immunoglobulin-type binding region does not comprisea ligand nor a receptor binding fragment thereof. In certain embodimentsof Embodiment Sets #1 to #20, the immunoglobulin-type binding regiondoes not comprise a chemokine or a TNF-related apoptosis-inducing ligand(TRAIL) nor a receptor binding fragment thereof. In certain embodimentsof Embodiment Sets #1 to #20, the binding region does not comprise ahuman CC chemokine nor a receptor binding fragment thereof. In certainembodiments of Embodiment Sets #1 to #20, the binding region does notcomprise the human CC chemokine CCL2 (see Bose S et al., Arch Pharm Res36; 1039-50 (2013)). In certain embodiments of Embodiment Sets #1 to#20, the binding region does not comprise the human, CC chemokine CCL2,nor a receptor binding fragment thereof and a carboxy-terminal, Shigatoxin effector polypeptide consisting of amino acids 75-247 of StxA. Incertain embodiments of the cell-targeting molecule of the presentinvention, the binding region does not comprise the human, CC chemokineCCL2, nor a receptor binding fragment thereof, fused to acarboxy-terminal, Shiga toxin effector polypeptide consisting of aminoacids 75-247 of StxA (SEQ ID NO:2). In embodiments of Embodiment Sets #1to #20, the binding region does not comprise the human TRAIL nor areceptor binding fragment thereof.

In certain embodiments, the cell-targeting molecule of the presentinvention comprises or consists essentially of the polypeptide of anyone of SEQ ID NOs: 252-255, 259-278, and 288-748. In certainembodiments, the cell-targeting molecule of the present invention doesnot comprise SEQ ID NO:25 and/or SEQ ID NO:247. In certain embodiments,the cell-targeting molecule of the present invention does not compriseor consist essentially of SEQ ID NO:287.

In certain embodiments of Embodiment Sets #1 to #20, the cell-targetingmolecule of the present invention is limited by the proviso that thecell-targeting molecule of the present invention does not comprise orconsist essentially of the polypeptide shown in SEQ ID NO:287 for allvariations described above of Embodiment Sets #1 to #20.

The present invention also provides pharmaceutical compositionscomprising a cell-targeting molecule of the present invention and atleast one pharmaceutically acceptable excipient or carrier; and the useof such a cell-targeting molecule, or a composition comprising it, inmethods of the invention as further described herein. Certainembodiments of the present invention are pharmaceutical compositionscomprising any cell-targeting molecule of the present invention; and atleast one pharmaceutically acceptable excipient or carrier.

Among certain embodiments of the present invention is a diagnosticcomposition comprising any one of the above cell-targeting molecules ofthe present invention and a detection promoting agent for the collectionof information, such as diagnostically useful information about acell-type, tissue, organ, disease, disorder, condition, and/or patient.Certain further embodiments are cell-targeting molecules of the presentinvention wherein the detection promoting agent is a heterologousepitope-peptide cargo and the cell-targeting molecule comprises theheterologous epitope-peptide cargo.

Beyond the cell-targeting molecules and compositions of the presentinvention, polynucleotides capable of encoding a cell-targeting moleculeof the present invention, or a protein component thereof, are within thescope of the present invention, as well as expression vectors whichcomprise a polynucleotide of the invention and host cells comprising anexpression vector of the invention. Host cells comprising an expressionvector may be used, e.g., in methods for producing a cell-targetingmolecule of the present invention, or a protein component or fragmentthereof, by recombinant expression.

The present invention also encompasses any composition of matter of thepresent invention which is immobilized on a solid substrate. Sucharrangements of the compositions of matter of the present invention maybe utilized, e.g., in methods of screening molecules as describedherein.

Among certain embodiments of the present invention is a method ofdelivering into a cell a CD8+ T-cell epitope-peptide cargo capable ofbeing presented by a MHC class I molecule of the cell, the methodcomprising the step of contacting the cell with the cell-targetingmolecule of the present invention and/or a composition thereof (e.g., apharmaceutical or diagnostic composition of the present invention).

Among certain embodiments of the present invention is a method ofinducing a cell to present an exogenously administered CD8+ T-cellepitope-peptide cargo complexed to a MHC class I molecule, the methodcomprising the step of contacting the cell, either in vitro or in vivo,with the cell-targeting molecule of the present invention, whichcomprises the CD8+ T-cell epitope, and/or a composition thereof (e.g., apharmaceutical or diagnostic composition of the present inventioncomprising such a cell-targeting molecule of the present invention).

Among certain embodiments of the present invention is a method ofinducing an immune cell-mediated response to target cell via a CD8+T-cell epitope MHC class I molecule complex, the method comprising thestep of contacting the target cell either in vitro or in vivo, with thecell-targeting molecule of the present invention, which comprises theCD8+ T-cell epitope as a cargo, and/or a composition thereof (e.g., apharmaceutical or diagnostic composition of the present inventioncomprising such a cell-targeting molecule of the present invention). Forcertain further embodiments, the immune response is selected from thegroup consisting: CD8+ immune cell secretion of a cytokine(s), cytotoxicT lymphocyte-(CTL) induced growth arrest in the target cell, CTL-inducednecrosis of the target cell, CTL-induced apoptosis of the target cell,immune cell-mediated cell killing of a cell other than the target cell.

Among certain embodiments of the present invention is a method ofcausing intercellular engagement of a CD8+ immune cell with a targetcell, the method comprises the step of contacting the target cell withthe cell-targeting molecule of the present invention in the presence ofa CD8+ immune cell or with the subsequent step of contacting the targetcell with one or more CD8+ immune cells. For certain embodiments, thecontacting step occurs in vitro. For certain other embodiments, thecontacting step occurs in vivo, such as, e.g., by administering thecell-targeting molecule to a chordate, vertebrate, and/or mammal. Forcertain embodiments, the intercellular engagement occurs in vitro. Forcertain embodiments, the intercellular engagement occurs in vivo.

Among certain embodiments of the present invention is a compositioncomprising a cell-targeting molecule of the present invention for“seeding” a tissue locus within a chordate.

For certain embodiments, a method of the present invention is for“seeding” a tissue locus within a chordate, the method comprising thestep of: administering to the chordate a cell-targeting molecule of thepresent invention, a pharmaceutical composition of the presentinvention, and/or a diagnostic composition of the present invention. Forcertain further embodiments, the method is for “seeding” a tissue locuswithin a chordate which comprises a malignant, diseased, and/or inflamedtissue. For certain further embodiments, the method is for “seeding” atissue locus within a chordate which comprises the tissue selected fromthe group consisting of: diseased tissue, tumor mass, cancerous growth,tumor, infected tissue, or abnormal cellular mass. For certainembodiments, the method for “seeding” a tissue locus within a chordatecomprises the step of: administering to the chordate a cell-targetingmolecule of the present invention comprising the heterologous, CD8+T-cell epitope-peptide cargo selected from the group consisting of:peptides not natively presented by the target cells of thecell-targeting molecule in MHC class I complexes, peptides not nativelypresent within any protein expressed by the target cell, peptides notnatively present within the transcriptome and/or proteome of the targetcell, peptides not natively present in the extracellularmicroenvironment of the site to be seeded, and peptides not nativelypresent in the tumor mass or infected tissue site to be targeted.

Additionally, the present invention provides methods of killing acell(s) comprising the step of contacting a cell(s) with acell-targeting molecule of the present invention or a pharmaceuticalcomposition comprising a cell-targeting molecule of the invention. Forcertain embodiments, the step of contacting the cell(s) occurs in vitro.For certain other embodiments, the step of contacting the cell(s) occursin vivo. For further embodiments of the cell-killing methods, the methodis capable of selectively killing cell(s) and/or cell-typespreferentially over other cell(s) and/or cell-types when contacting amixture of cells which differ with respect to the extracellular presenceand/or expression level of an extracellular target biomolecule of thebinding region of the cell-targeting molecule.

The present invention further provides methods of treating diseases,disorders, and/or conditions in patients in need thereof comprising thestep of administering to a patient in need thereof a therapeuticallyeffective amount of a composition comprising a cell-targeting moleculeor pharmaceutical composition of the present invention. For certainembodiments, the disease, disorder, or condition to be treated usingthis method of the invention is selected from: a cancer, tumor, growthabnormality, immune disorder, or microbial infection. For certainembodiments of this method, the cancer to be treated is selected fromthe group consisting of, bone cancer, breast cancer, central/peripheralnervous system cancer, gastrointestinal cancer, germ cell cancer,glandular cancer, head-neck cancer, hematological cancer, kidney-urinarytract cancer, liver cancer, lung/pleura cancer, prostate cancer,sarcoma, skin cancer, and uterine cancer. For certain embodiments ofthis method, the immune disorder to be treated is an immune disorderassociated with a disease selected from the group consisting of:amyloidosis, ankylosing spondylitis, asthma, Crohn's disease, diabetes,graft rejection, graft-versus-host disease, Hashimoto's thyroiditis,hemolytic uremic syndrome, HIV-related diseases, lupus erythematosus,multiple sclerosis, polyarteritis nodosa, polyarthritis, psoriasis,psoriatic arthritis, rheumatoid arthritis, scleroderma, septic shock,Sjögren's syndrome, ulcerative colitis, and vasculitis.

Among certain embodiments of the present invention is a compositioncomprising a cell-targeting molecule of the present invention for thetreatment or prevention of a cancer, tumor, growth abnormality, immunedisorder, or microbial infection. Among certain embodiments of thepresent invention is the use of a composition of matter of the presentinvention in the manufacture of a medicament for the treatment orprevention of a cancer, tumor, growth abnormality, immune disorder, ormicrobial infection.

The use of any composition of the present invention for the treatment orprevention of a cancer, tumor, growth abnormality, and/or immunedisorder is within the scope of the present invention.

Certain embodiments of the present invention include a method oftreating cancer in a patient using immunotherapy, the method comprisingthe step of administering to the patient in need thereof thecell-targeting molecule and/or pharmaceutical composition of the presentinvention.

The use of any composition of matter of the present invention for thetreatment or prevention of a cancer, tumor, growth abnormality, and/orimmune disorder is within the scope of the present invention. Amongcertain embodiments of the present invention is a cell-targetingmolecule of the present invention and/or a pharmaceutical compositionthereof for the treatment or prevention of a cancer, tumor, growthabnormality, immune disorder, and/or microbial infection. Among certainembodiments of the present invention is the use of a cell-targetingmolecule of the present invention and/or pharmaceutical compositionthereof in the manufacture of a medicament for the treatment orprevention of a cancer, tumor, growth abnormality, immune disorder, ormicrobial infection.

Among certain embodiments of the present invention is a compositioncomprising a cell-targeting molecule of the present invention for thedelivery of one or more additional exogenous materials into a cellphysically coupled with an extracellular target biomolecule of thebinding region of the cell-targeting molecule of the present invention.Certain embodiments of the cell-targeting molecules of the presentinvention may be used to deliver one or more additional exogenousmaterials into a cell physically coupled with an extracellular targetbiomolecule of the binding region of the cell-targeting molecule of thepresent invention. Additionally, the present invention provides a methodfor delivering exogenous material to the inside of a cell(s) comprisingcontacting the cell(s), either in vitro or in vivo, with acell-targeting molecule, pharmaceutical composition, and/or diagnosticcomposition of the present invention. The present invention furtherprovides a method for delivering exogenous material to the inside of acell(s) in a patient in need thereof the method comprising the step ofadministering to the patient a cell-targeting molecule of the presentinvention, wherein the target cell(s) is physically coupled with anextracellular target biomolecule of the binding region of thecell-targeting molecule of the present invention.

The use of any composition of the present invention (e.g. acell-targeting molecule, a pharmaceutical composition, or diagnosticcomposition) for the diagnosis, prognosis, and/or characterization of adisease, disorder, and/or condition is within the scope of the presentinvention.

Among certain embodiments of the present invention is the method ofdetecting a cell using a cell-targeting molecule and/or diagnosticcomposition of the invention comprising the steps of contacting a cellwith said cell-targeting molecule and/or diagnostic composition anddetecting the presence of said cell-targeting molecule and/or diagnosticcomposition. For certain embodiments, the step of contacting the cell(s)occurs in vitro. For certain embodiments, the step of contacting thecell(s) occurs in vivo. For certain embodiments, the step of detectingthe cell(s) occurs in vitro. For certain embodiments, the step ofdetecting the cell(s) occurs in vivo.

For example, a diagnostic composition of the invention may be used todetect a cell in vivo by administering to a chordate subject acomposition comprising cell-targeting molecule of the present inventionwhich comprises a detection promoting agent and then detecting thepresence of the cell-targeting molecule of the present invention and/orthe heterologous, CD8+ T-cell epitope-peptide cargo either in vitro orin vivo.

Certain embodiments of the cell-targeting molecules of the presentinvention may be utilized for the delivery of additional exogenousmaterial into a cell physically coupled with an extracellular targetbiomolecule of the cell-targeting molecule of the invention.Additionally, the present invention provides a method for deliveringexogenous material to the inside of a cell(s) comprising contacting thecell(s), either in vitro or in vivo, with a cell-targeting molecule,pharmaceutical composition, and/or diagnostic composition of the presentinvention. The present invention further provides a method fordelivering exogenous material to the inside of a cell(s) in a patient,the method comprising the step of administering to the patient acell-targeting molecule of the present invention (with or withoutcytotoxic activity), wherein the target cell(s) is physically coupledwith an extracellular target biomolecule of the cell-targeting molecule.

Among certain embodiments of the present invention is a method ofdelivering into a cell a T-cell epitope-peptide cargo capable of beingpresented by a MHC class I molecule of the cell, the method comprisingthe step of contacting the cell with the cell-targeting molecule of thepresent invention which is associated with a heterologous, T-cellepitope-peptide cargo and/or a composition thereof (e.g., apharmaceutical or diagnostic composition of the present invention).

Among certain embodiments of the present invention is a method for“seeding” a tissue locus within a chordate, the method comprising thestep of: administering to the chordate a cell-targeting molecule of thepresent invention, a pharmaceutical composition of the presentinvention, and/or a diagnostic composition of the present invention. Incertain further embodiments, the methods of the invention for “seeding”a tissue locus are for “seeding” a tissue locus which comprises amalignant, diseased, or inflamed tissue. In certain further embodiments,the methods of the invention for “seeding” a tissue locus are for“seeding” a tissue locus which comprises the tissue selected from thegroup consisting of: diseased tissue, tumor mass, cancerous growth,tumor, infected tissue, or abnormal cellular mass. In certain furtherembodiments, the methods of the invention for “seeding” a tissue locuscomprises administering to the chordate the cell-targeting molecule ofthe invention, the pharmaceutical composition of the invention, or thediagnostic composition of the invention comprising the heterologous,T-cell epitope-peptide cargo selected from the group consisting of:peptides not natively presented by the target cells of thecell-targeting molecule in MHC class I complexes, peptides not nativelypresent within any protein expressed by the target cell, peptides notnatively present within the proteome of the target cell, peptides notnatively present in the extracellular microenvironment of the site to beseeded, and peptides not natively present in the tumor mass or infectedtissue site to be targeted.

The use of any composition of matter of the present invention for thediagnosis, prognosis, and/or characterization of a disease, disorder,and/or condition is within the scope of the present invention. Amongcertain embodiments of the present invention is a method of using acell-targeting molecule of the present invention comprising a detectionpromoting agent and/or composition of the invention (e.g. a diagnosticcomposition) for the collection of information useful in the diagnosis,prognosis, or characterization of a disease, disorder, or condition.Among certain embodiments of the present invention is the method ofdetecting a cell (or subcellular compartment thereof) using acell-targeting molecule and/or diagnostic composition of the presentinvention, the method comprising the steps of contacting a cell with thecell-targeting molecule and/or diagnostic composition and detecting thepresence of said cell-targeting molecule and/or diagnostic composition.In certain embodiments, the step of contacting the cell(s) occurs invitro. In certain embodiments, the step of contacting the cell(s) occursin vivo. In certain embodiments, the step of detecting the cell(s)occurs in vitro. In certain embodiments, the step of detecting thecell(s) occurs in vivo. In certain further embodiments, the methodinvolves the detection of the location of the cell-targeting molecule inan organism using one or more imaging procedures after theadministration of the cell-targeting molecule to said organism. Forexample, cell-targeting molecules of the invention which incorporatedetection promoting agents as described herein may be used tocharacterize diseases as potentially treatable by a relatedpharmaceutical composition of the present invention. For example,certain cell-targeting molecules of the present invention andcompositions thereof (e.g. pharmaceutical compositions and diagnosticcompositions of the present invention), and methods of the presentinvention may be used to determine if a patient belongs to a group thatresponds to a pharmaceutical composition of the present invention. Forexample, certain cell-targeting molecules of the present invention andcompositions thereof may be used to identify cells which present adelivered heterologous epitope-peptide cargo on a cellular surfaceand/or to identify subjects containing cells which present aheterologous epitope-peptide cargo delivered by a cell-targetingmolecule of the present invention.

Among certain embodiments of the present invention is a method ofproducing a molecule of the present invention, the method comprising thestep of purifying the molecule of the invention or a polypeptidecomponent of thereof using a bacterial cell-wall protein domaininteraction, such as, e.g., protein L from P. magnus or derivatives andbinding domain fragments thereof. In certain further embodiments, thepurifying step of the method involves the Shiga toxin effectorpolypeptide comprising or consisting essentially of any one of thepolypeptides shown in SEQ ID NOs: 29-38.

Among certain embodiments of the present invention are kits comprising acomposition of matter of the invention, and optionally, instructions foruse, additional reagent(s), and/or pharmaceutical delivery device(s).The kit may further comprise reagents and other tools for detecting acell type (e.g. a tumor cell) in a sample or in a subject.

These and other features, aspects and advantages of the presentinvention will become better understood with regard to the followingdescription, appended claims, and accompanying figures. Theaforementioned elements of the invention may be individually combined orremoved freely in order to make other embodiments of the invention,without any statement to object to such combination or removalhereinafter.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A and FIG. 1B depict exemplary, cell-targeting moleculescomprising a heterologous, CD8+ T-cell epitope-peptide cargo (shown as adiamond shape). These exemplary cell-targeting molecules each comprise aShiga toxin effector polypeptide having a combination of features:de-immunizing mutations (shown as a horizontally striped region), anembedded, heterologous, CD8+ T-cell epitope (shown as a verticallystriped region), and sometimes a disrupted or missing protease site(shown with a dashed line). The “N” and “C” denote an amino-terminus andcarboxy-terminus, respectively, of a polypeptide component of acell-targeting molecule. These exemplary cell-targeting moleculessometimes comprise a Shiga toxin effector polypeptide having the thirdfeature of a disrupted furin-cleavage site at the carboxy-terminus of anA1 fragment derived region depicted with a dashed, vertical, gray line.

The depictions of exemplary molecules in FIG. 1A and FIG. 1B are forillustrative purposes of certain, general arrangements of the structuralfeatures of a limited set of embodiments of the present invention. It isto be understood that these exemplary molecules do not intend, norshould any be construed, to be wholly definitive as to the arrangementof any structural features and/or components of a molecule of thepresent invention. The relative size, location, or number of featuresshown in the schematics of FIG. 1A and FIG. 1B have been simplified. Forexample, the relative positions of embedded, heterologous epitopes anddisruptions of an endogenous, epitope regions are not fixed. Similarly,the total numbers of embedded, heterologous epitopes and disruptions ofan endogenous, epitope regions are not fixed. Certain embodiments of themolecules of the present invention comprise a plurality of disrupted,endogenous, epitope regions in a single, Shiga toxin effectorpolypeptide, such as, e.g., disruptions of four, five, six, seven,eight, nine, or more regions; wherein these disrupted, endogenous,epitope regions may be distributed throughout the Shiga toxin effectorpolypeptide, including disruptions which overlap with or are within thefurin-cleavage motif of the carboxy-terminus region of a Shiga toxin A1fragment derived region (see e.g. WO 2016/196344). Certain embodimentsof the present invention comprise disruptions of endogenous, epitoperegions which are carboxy-terminal to the carboxy-terminus of the Shigatoxin A1 fragment, or a derivative thereof such as, e.g. at a positioncarboxy-terminal to any disrupted furin-cleavage site motif. Theschematics in FIG. 1A and FIG. 1B are not intended to accurately portrayany information regarding the relative sizes of molecular structures inany embodiment of the present invention.

FIG. 2 graphically shows that the exemplary cell-targeting moleculeSLT-1A-DI-FR::scFv1::C2 (SEQ ID NO:252) exhibited cytotoxicity to two,different cell-types comparable to a “control” cell-targeting moleculeSLT-1A-DI-FR::scFv-1 (SEQ ID NO:258). The percent viability of targetpositive cells for each cell type was plotted over the logarithm to base10 of the cell-targeting molecule concentration administered to therespective cells.

FIG. 3 graphically shows cell-surface presentation of a cell-targetingmolecule delivered, heterologous, CD8+ T-cell epitope-peptide complexedwith MHC class I molecule by a target positive cancer cell as comparedto a negative control. FIG. 3 shows overlays of the results of aTCR-STAR™ assay, flow cytometric analysis of sets of cells treatedeither with the exemplary cell-targeting molecule of the presentinvention SLT-A-DI-FR::scFv1::C2 (SEQ ID NO:252) or a negative control,the cell-targeting molecule SLT-1A-DI-FR::scFv1 (SEQ ID NO:258), whichlacks any C2 epitope-peptide cargo. The FACS cell count of targetpositive cells was plotted over the light signal from PE-STAR™ multimerreagent in relative light units (RLU) representing the presence ofcell-surface, MHC class I molecule (human HLA-A2) displayed C2epitope-peptide (SEQ ID NO:21) complexes. Target positive cells treatedwith the exemplary cell-targeting molecule of the present inventionSLT-1A-DI-FR::scFv1::C2 (SEQ ID NO:252) displayed the C2 epitope-peptide(SEQ ID NO:21) complexed to MHC class I molecules on their cell surfaces(upper graph), whereas target positive cells treated with the parentalcell-targeting molecule SLT-1A-DI-FR::scFv1 (SEQ ID NO:258) did notdisplay the C2 epitope-peptide (SEQ ID NO:21) on a cell surface (lowergraph).

FIG. 4 graphically shows the sizes and proportions of molecules presentin a sample preparation of SLT-1A-DI-FR::scFv1::C2 (SEQ ID NO:252)analyzed by size exclusion chromatography (SEC). For the SEC analysis,the absorbance of ultraviolet light at a wavelength of 280 nanometers(nm) of the material eluted after flowing through a SEC column wasplotted in milli-absorbance units (mAU) over the fraction volume inmilliliters (mL). Software was used to identify individual peaks in the280 nm trace and the fraction volume of each peak's maximum absorbanceof ultraviolet light at 280 nm.

FIG. 5 shows a Coomassie-stained, sodium dodecyl sulfate, polyacrylamidegel (SDS-PAGE) after electrophoresis of sample preparation ofSLT-1A-DI-FR::scFv1:C2 (SEQ ID NO:252) prepared for gel-loading underreducing conditions. FIG. 5 shows that size of reducedSLT-1A-DI-FR::scFv1::C2 (SEQ ID NO:252) was about 55 kiloDaltons (kDa).

FIG. 6 graphically shows fusing a heterologous, CD8+ T-cellepitope-peptide to a Shiga toxin A Subunit derived, cell-targetingmolecule did not significantly impair the cytotoxic activity of thecell-targeting molecule toward target positive cells. The percentviability of cells was plotted over the logarithm to base 10 of theprotein concentration. FIG. 6 graphically shows the results of acell-kill assay where SLT-1A::scFv1::C2 (SEQ ID NO:278) exhibitedcytotoxicity similar to the cytotoxicity of the parental cell-targetingmolecule SLT-1A::scFv1 (SEQ ID NO:280), which lacked any heterologous,CD8+ T-cell epitope-peptide.

FIG. 7 graphically shows the results of a cell-kill assay where thecytotoxic activity of the exemplary cell-targeting moleculeSLT-1A::scFv1::C2 (SEQ ID NO:278) was specific to target positive cellsover a certain concentration range. The percent viability of cells wasplotted over the logarithm to base 10 of the protein concentration.Cells negative for cell-surface expression of a target biomolecule ofthe binding region scFv2 were not killed (approximately 100% cellviability) by SLT-1A::scFv1::C2 (SEQ ID NO:278) over the moleculeconcentration range used to accurately measure the CD₅₀ value ofSLT-1A::scFv1::C2 (SEQ ID NO:278) toward target positive cells and asshown in FIG. 6.

FIG. 8 graphically shows cell-surface presentation of a cell-targetingmolecule delivered, heterologous, CD8+ T-cell epitope-peptide complexedwith MHC class I molecule by a target positive cancer cell as comparedto a negative control. FIG. 8 shows overlays of the results of aTCR-STAR™ assay, flow cytometric analysis of sets of cells treatedeither with a negative control, the cell-targeting moleculeSLT-1A::scFv1::C2 (SEQ ID NO:278), or the cell-targeting moleculeSLT-1A::scFv2 (SEQ ID NO:281). The fluorescence-activated cell sorting(FACS) flow cytometry cell count of target positive cells was plottedover the light signal from PE-STAR™ multimer reagent in relative lightunits (RLU) representing the presence of cell-surface, MHC class Imolecule (human HLA-A2) displayed C2 epitope-peptide (SEQ ID NO:21)complexes. Target positive cells treated with the exemplarycell-targeting molecule of the present invention SLT-1A::scFv1::C2 (SEQID NO:278) displayed the C2 epitope-peptide (SEQ ID NO:21) complexed toMHC class I molecules on their cell surfaces (upper graph), whereastarget positive cells treated with the related cell-targeting moleculeSLT-1A::scFv2 (SEQ ID NO:281) did not display the C2 epitope-peptide(SEQ ID NO:21) on a cell surface (lower graph).

FIG. 9 graphically shows cell-surface presentation of a cell-targetingmolecule delivered, heterologous, CD8+ T-cell epitope-peptide complexedwith MHC class I molecule by a target positive cancer cell as comparedto negative controls. In FIG. 9, the indexed, mean, fluorescentintensity (“iMFI,” the fluorescence of the positive populationmultiplied by the percent positive) of the PE-STAR™ multimer reagent inRLU corresponding to the sets of cells receiving the differenttreatments was graphed. FIG. 9 shows the results of a TCR-STAR Assay™,flow cytometric analysis of cells treated with either an exogenous C2peptide (SEQ ID NO:21) control, “inactive SLT-1A::scFv” (SEQ ID NO:282),or the cell-targeting molecule “inactive SLT-1A::scFv2::C2” (SEQ IDNO:270). Exogenously administered C2 peptide ((SEQ ID NO:21), as above)combined with a Peptide Loading Enhancer (“PLE,” Altor Bioscience Corp.,Miramar, Fla., U.S.). The C2 peptide (SEQ ID NO:21) combined withPeptide Loading Enhancer (PLE) treatment provides a positive controlwhere exogenously administered C2 peptide (SEQ ID NO:21) may be loadedonto cell-surface MHC class I molecules without ever entering a cell.Target positive cells treated with the exemplary cell-targeting moleculeof the present invention “inactive SLT-1A::scFv2::C2” (SEQ ID NO:270)displayed the C2 epitope-peptide (SEQ ID NO:21) complexed to MI-IC classI molecules on their cell surfaces, whereas the same cells treated withonly exogenous C2 epitope-peptide (SEQ ID NO:21) or the parentalcell-targeting molecule “inactive SLT-1A::scFv2” (SEQ ID NO:282) did notdisplay the C2 epitope-peptide (SEQ ID NO:21) on a cell surface.

FIG. 10 graphically shows cell-surface presentation of a cell-targetingmolecule delivered, heterologous, CD8+ T-cell epitope-peptide complexedwith MHC class I molecule by a target positive cancer cell for differentincubation times (4 hours or 16 hours) as compared to a negativecontrol. FIG. 10 shows overlays of the results of a TCR-STAR™ assay,flow cytometric analysis of sets of cells treated either with thecell-targeting molecule SLT-1A::scFv1::C2 (SEQ ID NO:278) or a negativecontrol. The FACS cell count of target positive cells was plotted overthe light signal from PE-STAR™ multimer reagent in relative light units(RLU) representing the presence of cell-surface, MHC class I molecule(human HLA-A2) displayed C2 epitope-peptide (SEQ ID NO:21) complexes.Target positive cells treated with the exemplary cell-targeting moleculeof the present invention SLT-1A::scFv1::C2 (SEQ ID NO:278) displayed theC2 epitope-peptide (SEQ ID NO:21) complexed to MHC class I molecules ontheir cell surfaces after either a 4-hour (4 hrs) (upper graph) or16-hour (16 hrs) (lower graph) incubation duration.

FIG. 11 graphically shows cell-surface presentation of a cell-targetingmolecule delivered, heterologous, CD8+ T-cell epitope-peptide complexedwith MHC class I molecule by a target positive cancer cell as comparedto a negative control. FIG. 11 shows overlays of the results of aTCR-STAR™ assay, flow cytometric analysis of sets of cells treatedeither with a negative control, the cell-targeting moleculeSLT-1A::scFv5::C2 (SEQ ID NO:274), or the cell-targeting moleculeSLT-1A::scFv5 (SEQ ID NO:283). The FACS cell count of target positivecells was plotted over the light signal from PE-STAR™ multimer reagentin relative light units (RLU) representing the presence of cell-surface,MHC class I molecule (human HLA-A2) displayed C2 epitope-peptide (SEQ IDNO:21) complexes. Target positive cells treated with the exemplarycell-targeting molecule of the present invention SLT-1A::scFv5::C2 (SEQID NO:274) displayed the C2 epitope-peptide (SEQ ID NO:21) complexed toMHC class I molecules on their cell surfaces (upper graph), whereastarget positive cells treated with the parental cell-targeting moleculeSLT-1A::scFv5 (SEQ ID NO:283) did not display the C2 epitope-peptide(SEQ ID NO:21) on a cell surface (lower graph).

FIG. 12 graphically shows cell-surface presentation of a cell-targetingmolecule delivered, heterologous, CD8+ T-cell epitope-peptide complexedwith MHC class I molecule by a target positive cancer cell as comparedto a negative control. FIG. 12 shows overlays of the results of aTCR-STAR™ assay, flow cytometric analysis of sets of cells treatedeither with a negative control, the cell-targeting moleculeSLT-1A::scFv7::C2 (SEQ ID NO:277), or the cell-targeting moleculeSLT-1A::scFv7 (SEQ ID NO:286). The FACS cell count of target positivecells was plotted over the light signal from PE-STAR™ multimer reagentin relative light units (RLU) representing the presence of cell-surface,MHC class I molecule (human HLA-A2) displayed C2 epitope-peptide (SEQ IDNO:21) complexes. Target positive cells treated with the exemplarycell-targeting molecule of the present invention SLT-1A::scFv7::C2 (SEQID NO:277) displayed the C2 epitope-peptide (SEQ ID NO:21) complexed toMHC class I molecules on their cell surfaces (upper graph), whereastarget positive cells treated with the parental cell-targeting moleculeSLT-1A::scFv7 (SEQ ID NO:286) did not display the C2 epitope-peptide(SEQ ID NO:21) on a cell surface (lower graph).

FIG. 13 graphically shows the results from an Interferon Gamma ELIspotassay with the number of spots, or secreting cells, plotted for eachcondition tested. For each sample, target positive human cancer cellswere pretreated with a cell-targeting molecule or negative control and,then, incubated with human PBMCs (HLA-A2 serotype) for 24 hours beforeperforming the ELISPOT assay. FIG. 13 shows that target positive cellstreated with the exemplary cell-targeting molecule of the presentinvention “inactive SLT-1A::scFv2::C2” (SEQ ID NO:270) promoted cytokinesecretion by PBMCs, whereas treatment with the parental cell-targetingmolecule “inactive SLT-1A::scFv2” (SEQ ID NO:282) resulted only in abackground level of interferon-y secretion at about the same level asthe negative control treatment of “buffer only.” The notation “Targetpositive Cell Line G+ PBMCs” indicates that all the samples showninvolved a coculture of tumor cells of cell line G with PBMCs.

FIG. 14 graphically shows the results from an intercellular T lymphocyte(T-cell) activation assay with luciferase activity plotted in RLU foreach condition tested. For each sample, target positive human cancercells were pretreated with a cell-targeting molecule or negative controland, then, incubated for 18 hours with human J76 T-cells expressing ahuman T-cell receptor (TCR) that specifically recognizes cell-surfacepresented, human MHC class I molecule (HLA-A2) F2 epitope (SEQ ID NO:25)complexes, and comprising a nuclear factor of activated T-cells (NFAT)transcriptional response element driving luciferase expression(luciferase-reporter-transfected). Luciferase activity in the samples,indicating NFAT activity in the T-cells, was measured using the One-Glo™Luciferase Assay System reagent according the manufacturer'sinstructions. FIG. 14 shows that target positive tumor cells pretreatedwith the exemplary cell-targeting molecule of the present invention“inactive SLT-1A::scFv6::F2” (SEQ ID NO:276) stimulated anintermolecular response in the form of T-cell activation via TCRrecognition and NFAT signaling: whereas, treatment of target positivecells with the parental cell-targeting molecule “inactive SLT-1A::scFv6”(SEQ ID NO:285) resulted only in the background level of light signal atabout the same level as the negative control treatment of “buffer only.”The notation “Target positive Cell Line F+ Reporter T cells” indicatesthat all the samples shown involved a coculture of tumor cells of cellline G with the luciferase-reporter transfected J76 T-cells (HLA-A2serotype) as described above.

FIG. 15 graphically shows the results from an IFN-γ ELISA assay withdata from the “buffer only” sample in black and from the cell-targetingmolecule treated sample in grey. Along the Y-axis is the quantificationof IFN-γ present in the supernatants from a coculture experimentreported in picograms per mL (pg-mL). The cells of the cocultureconsisted of human PBMCs enriched for C2-restricted PBMCs and humantumor cells. The tumor cells used were of the cell line I and werepretreated with “inactive SLT-1A-DI-4::scFv6::(C2)3” (SEQ ID NO:253).FIG. 15 shows that coculture of PBMCs with target positive tumor cellsafter incubation with the cell targeting molecule “inactiveSLT-1A-DI-4::scFv6::(C2)3” (SEQ ID NO:253) but not with “buffer only”results in the induction of human IFN-γ secretion indicative ofactivated T-cells among the PBMCs.

FIG. 16 graphically shows the results from a CellTiter-Glo® LuminescentCell Viability assay of a coculture experiment with data from the“buffer only” sample in black and from the cell-targeting moleculetreated sample in grey. Along the Y-axis is the quantification ofadherent-cell viability expressed as a percentage of the “buffer only”negative control in RLU. The cells of the coculture consisted of humanPBMCs enriched for C2-restricted PBMCs and human tumor cells. The tumorcells used were of the cell line I and were pretreated with “inactiveSLT-1A-DI-4::scFv6::(C2)3” (SEQ ID NO:253). FIG. 16 shows that coculturePBMCs with target positive tumor cells after incubation with the celltargeting molecule “inactive SLT-1A-DI-4::scFv6::(C2)3” (SEQ ID NO:253)but not with “buffer only” results in tumor cell-killing as demonstratedby the large reduction in the viability percentage of the adherent tumorcells.

FIG. 17 graphically plots the number of immune cells clustered duringdifferent time-points of a 140-hour coculture experiment with data fromthe “buffer only” sample in black and from the cell-targeting moleculetreated sample in grey. The cells were counted using the IncuCyte® S3Live-Cell imaging assay performed as described herein. The cells of thecoculture consisted of human PBMCs enriched for C2-restricted PBMCs andhuman tumor cells. The tumor cells used were of the cell line I and werepretreated with “inactive SLT-1A-DI-4::scFv6::(C2)3” (SEQ ID NO:253).The quantity cells involved and the quantity of PBMC clusters of over100 microns are indicative of immune cell activation occurring in thecoculture. The quantities of cells and clusters over time (thepersistency of immune cell clustering behavior) is also indicativeimmune cell activation occurring in the coculture.

FIG. 18 graphically shows the results from IFN-γ ELISA assays with datafrom three-different tumor cell types treated with three differentconcentrations of an exemplary cell-targeting molecule of the presentinvention (SEQ ID NO:254) (shown in grey) and data from a controltreatment using a cell-targeting molecule lacking any C2 epitope-peptidecargo for delivery (SEQ ID NO:256) (shown in black). Along the Y-axis isthe quantification of IFN-γ present in the supernatants from cocultureexperiments reported in pg/mL. The x-axis shows four differentexperimental conditions: no PBMC-coculture, “buffer only” treatment oftumor cells before coculture, treatment of tumor cells with 100 nM ofmolecule, and treatment with tumor cells with 500 nM of molecule. Thecells of the coculture consisted of human PBMCs enriched forC2-restricted PBMCs and human tumor cells. The tumor cells used were ofthe cell line I, J, or K and were pretreated with either “inactiveSLT-1A-DI-1::scFv8::C2” (SEQ ID NO:254) or “inactive SLT-1A-DI-1::scFv8”(SEQ ID NO:256). As the concentration of exemplary cell-targetingmolecule of the present invention in the treatment goes up from zero to100, and 500 nM, the quantity of secreted human IFN-γ in the coculturegoes up (e.g., to above about 400 or 600 pg/mL). The results show thatneither “buffer only” or the closely related reference cell-targetingmolecule “inactive SLT-1A-DI-1::scFv8” (SEQ ID NO:256) lacking the of C2epitope-peptide (SEQ ID NO:21) was capable of inducing IFN-γ secretionafter 48 hours post-coculture. The notation “+ PBMCs” indicates thesample shown involved a coculture of tumor cells and PBMCs.

FIG. 19 graphically shows the results from CellTiter-Glo® LuminescentCell Viability assays of coculture experiments with data fromthree-different tumor cell types treated with three differentconcentrations of an exemplary cell-targeting molecule of the presentinvention (SEQ ID NO:254) (shown in grey) and data from a controltreatment using a cell-targeting molecule lacking any C2 epitope-peptidecargo for delivery (SEQ ID NO:256) (shown in black). Along the Y-axis isthe quantification of adherent-cell viability in RLU. The x-axis showsfour different experimental conditions: no PBMC-coculture, “buffer only”treatment of tumor cells before coculture, treatment of tumor cells with100 nM of molecule, and treatment with tumor cells with 500 nM ofmolecule. The cells of the coculture consisted of human PBMCs enrichedfor C2-restricted PBMCs and human tumor cells. The tumor cells used wereof the cell line I, J, or K and were pretreated with either “inactiveSLT-1A-DI-1::scFv8::C2” (SEQ ID NO:254) or “inactive SLT-1A-DI-1::scFv8”(SEQ ID NO:256). As the concentration of the exemplary cell-targetingmolecule of the present invention “inactive SLT-1A-DI-1::scFv8::C2” (SEQID NO:254) in the treatment goes up from zero to 100, and 500 nM, theadherent cell viability drops to below 50 percent. The results show thatneither “buffer only” or the closely related reference cell-targetingmolecule “inactive SLT-1A-DI-FR::scFv8” (SEQ ID NO:256) lacking the ofC2 epitope-peptide (SEQ ID NO:21) was capable of causing significanttarget cell death during 96 hours of coculture. The notation “+ PBMCs”indicates the sample shown involved a coculture of tumor cells andPBMCs.

FIG. 20 graphically shows the results from IFN-γ ELISA andCellTiter-Glo® Luminescent Cell Viability assays of cocultureexperiments with pretreatment of tumor cells with an exemplarycell-targeting molecule of the present invention (SEQ ID NO:255) shownin grey and data from the control treatment using a catalytically-activecell-targeting molecule lacking any C2 epitope-peptide cargo fordelivery shown in black (SEQ ID NO:257). The cells of the cocultureconsisted of human PBMCs enriched for C2-restricted PBMCs and humantumor cells. The tumor cells used were of the cell line I and werepretreated with the catalytically-active SLT-1A-DI-1::scFv8::C2 (SEQ IDNO:255) or with the catalytically active control moleculeSLT-1A-DI-1::scFv8 (SEQ ID NO:257). For the graph on the left side, theY-axis shows the quantification of IFN-γ present in the supernatantsfrom the coculture experiments reported in pg/mL. For the graph on theright side, the Y-axis shows the quantification of adherent-cellviability in RLU. For both graphs, the x-axis shows four differentexperimental conditions: no PBMC-coculture, “buffer only” treatment oftumor cells before coculture, treatment of tumor cells with 30 nM ofmolecule, and treatment of tumor cells with 100 nM of molecule. As theconcentration of the exemplary cell-targeting molecule of the presentinvention SLT-1A-DI-1::scFv8::C2 (SEQ ID NO:255) in the treatment goesup from zero to and 100 nM, the quantity of secreted human IFN-γ in thecoculture goes up to above 200 pg/mL and the adherent cell viability isreduced more than treatment with a closely related, catalytically-activecell-targeting molecule SLT-1A-DI-1::scFv8 (SEQ ID NO:257) lacking anyC2 epitope-peptide (SEQ ID NO:21) cargo. The control molecule did notinduce any human IFN-γ secretion by the PBMCs in coculture and did notreduce tumor cell viability over time as much as the exemplary moleculeof the present invention did for a given molecule treatmentconcentration. The notation “+ PBMCs” indicates the sample showninvolved a coculture of tumor cells and PBMCs.

DETAILED DESCRIPTION

The present invention is described more fully hereinafter usingillustrative, non-limiting embodiments, and references to theaccompanying figures. This invention may, however, be embodied in manydifferent forms and should not be construed as to be limited to theembodiments set forth below. Rather, these embodiments are provided sothat this disclosure is thorough and conveys the scope of the inventionto those skilled in the art. In order that the present invention may bemore readily understood, certain terms are defined below. Additionaldefinitions may be found within the detailed description of theinvention.

As used in the specification and the appended claims, the terms “a,”“an” and “the” include both singular and the plural referents unless thecontext clearly dictates otherwise.

As used in the specification and the appended claims, the term “and/or”when referring to two species, A and B, means at least one of A and B.As used in the specification and the appended claims, the term “and/or”when referring to greater than two species, such as A, B, and C, meansat least one of A, B, or C, or at least one of any combination of A, B,or C (with each species in singular or multiple possibility).

Throughout this specification, the word “comprise” or variations such as“comprises” or “comprising” will be understood to imply the inclusion ofa stated integer (or components) or group of integers (or components),but not the exclusion of any other integer (or components) or group ofintegers (or components).

Throughout this specification, the term “including” is used to mean“including but not limited to.” “Including” and “including but notlimited to” are used interchangeably.

The term “amino acid residue” or “amino acid” includes reference to anamino acid that is incorporated into a protein, polypeptide, and/orpeptide. The term “polypeptide” includes any polymer of amino acids oramino acid residues. The term “polypeptide sequence” refers to a seriesof amino acids or amino acid residues which physically comprise apolypeptide. A “protein” is a macromolecule comprising one or morepolypeptides or polypeptide “chains.” A “peptide” is a small polypeptideof sizes less than about a total of 15 to 20 amino acid residues.

The term “amino acid sequence” refers to a series of amino acids oramino acid residues which physically comprise a peptide or polypeptidedepending on the length. Unless otherwise indicated, polypeptide andprotein sequences disclosed herein are written from left to rightrepresenting their order from an amino terminus to a carboxy terminus.

The terms “amino acid,” “amino acid residue,” “amino acid sequence,” orpolypeptide sequence include naturally occurring amino acids (includingL and D isosteriomers) and, unless otherwise limited, also include knownanalogs of natural amino acids that can function in a similar manner asthe common natural amino acids, such as selenocysteine, pyrrolysine,N-formylmethionine, gamma-carboxyglutamate, hydroxyprolinehypusine,pyroglutamic acid, and selenomethionine (see e.g. Young T, Schultz P, JBiol Chem 285; 11039-44 (2010); Davis L, Chin J, Nat Rev Mol Cell Biol13; 168-82 (2012); Bohike N, Budisa N. FEMS Microbiol Lett 35; 133-44(2014); Chin J, Annu Rev Biochem 83; 379-408 (2014); Nagata K et al.,Bioinformatics 30; 1681-9 (2014); Pott M et al., ACS Chem Biol 9:2815-22 (2014); Ho J et al., ACS Synth Biol 5: 163-71 (2016); Wang Y,Tsao M, Chembiochem 17: 2234-9 (2016)). The amino acids referred toherein are described by shorthand designations as follows in Table A:

TABLE A Amino Acid Nomenclature Name 3-letter 1-letter Alanine Ala AArginine Arg R Asparagine Asn N Aspartic Acid or Aspartate Asp DCysteine Cys C Glutamic Acid or Glutamate Glu E Glutamine Gln Q GlycineGly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys KMethionine Met M Phenylalanine Phe F Proline Pro P Serine Ser SThreonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

The phrase “conservative substitution” with regard to an amino acidresidue of a peptide, peptide region, polypeptide region, protein, ormolecule refers to a change in the amino acid composition of thepeptide, peptide region, polypeptide region, protein, or molecule thatdoes not substantially alter the function and structure of the overallpeptide, peptide region, polypeptide region, protein, or molecule (seeCreighton, Proteins: Structures and Molecular Properties (W. H. Freemanand Company, New York (2nd ed., 1992))).

For purposes of the present invention, the phrase “derived from” whenreferring to a polypeptide or polypeptide region means that thepolypeptide or polypeptide region comprises highly similar amino acidsequences originally found in a “parental” protein and which may nowcomprise certain amino acid residue additions, deletions, truncations,rearrangements, or other alterations relative to the originalpolypeptide or polypeptide region as long as a certain function(s) and astructure(s) of the “parental” molecule are substantially conserved. Theskilled worker will be able to identify a parental molecule from which apolypeptide or polypeptide region was derived using techniques known inthe art, e.g., protein sequence alignment software.

For purposes of the present invention, the terms “terminus.” “aminoterminus,” or “carboxy terminus” with regard to a polypeptide regionrefers to the regional boundaries of that region, regardless of whetheradditional amino acid residues are linked by peptide bonds outside ofthat region. In other words, the terminals of the polypeptide regionregardless of whether that region is fused to other peptides orpolypeptides. For example, a fusion protein comprising two proteinaceousregions, e.g., a binding region comprising a peptide or polypeptide anda Shiga toxin effector polypeptide, may have a Shiga toxin effectorpolypeptide region with a carboxy terminus ending at amino acid residue251 of the Shiga toxin effector polypeptide region despite a peptidebond involving residue 251 to an amino acid residue at position 252representing the beginning of another proteinaceous region, e.g., thebinding region. In this example, the carboxy terminus of the Shiga toxineffector polypeptide region refers to residue 251, which is not aterminus of the fusion protein but rather represents an internal,regional boundary. Thus, for polypeptide regions, the terms “terminus,”“amino terminus,” and “carboxy terminus” are used to refer to theboundaries of polypeptide regions, whether the boundary is a physicallyterminus or an internal, position embedded within a larger, continuouspolypeptide chain.

For purposes of the claimed invention and with regard to a Shiga toxinpolypeptide sequence or Shiga toxin derived polypeptide, the term“wild-type” generally refers to a naturally occurring, Shiga toxinprotein sequence(s) found in a living species, such as, e.g., apathogenic bacterium, wherein that Shiga toxin protein sequence(s) isone of the most frequently occurring variants. This is in contrast toinfrequently occurring Shiga toxin protein sequences that, while stillnaturally occurring, are found in less than one percent of individualorganisms of a given species when sampling a statistically powerfulnumber of naturally occurring individual organisms of that species whichcomprise at least one Shiga toxin protein variant. A clonal expansion ofa natural isolate outside its natural environment (regardless of whetherthe isolate is an organism or molecule comprising biological sequenceinformation) does not alter the naturally occurring requirement as longas the clonal expansion does not introduce new sequence variety notpresent in naturally occurring populations of that species and/or doesnot change the relative proportions of sequence variants to each other.

The terms “associated,” “associating,” “linked,” or “linking” withregard to the claimed invention refers to the state of two or morecomponents of a molecule being joined, attached, connected, or otherwisecoupled to form a single molecule or the act of making two moleculesassociated with each other to form a single molecule by creating anassociation, linkage, attachment, and/or any other connection betweenthe two molecules. For example, the term “linked” may refer to two ormore components associated by one or more atomic interactions such thata single molecule is formed and wherein the atomic interactions may becovalent and/or non-covalent. Non-limiting examples of covalentassociations between two components include peptide bonds andcysteine-cysteine disulfide bonds. Non-limiting examples of non-covalentassociations between two molecular components include ionic bonds.

For purposes of the present invention, the term “linked” refer to two ormore molecular components associated by one or more atomic interactionssuch that a single molecule is formed and wherein the atomicinteractions include at least one covalent bond. For purposes of thepresent invention, the term “linking” refers to the act of creating alinked molecule as described above.

For purposes of the present invention, the term “fused” refers to two ormore proteinaceous components associated by at least one covalent bondwhich is a peptide bond, regardless of whether the peptide bond involvesthe participation of a carbon atom of a carboxyl acid group or involvesanother carbon atom, such as, e.g., the α-carbon, β-carbon, γ-carbon,σ-carbon, etc. Non-limiting examples of two proteinaceous componentsfused together include, e.g., an amino acid, peptide, or polypeptidefused to a polypeptide via a peptide bond such that the resultingmolecule is a single, continuous polypeptide. For purposes of thepresent invention, the term “fusing” refers to the act of creating afused molecule as described above, such as, e.g., a fusion proteingenerated from the recombinant fusion of genetic regions which whentranslated produces a single proteinaceous molecule.

The symbol “::” means the polypeptide regions before and after it arephysically linked together to form a continuous polypeptide.

Throughout this specification, the term “bispecific” will be understoodto include molecules which bind different extracellular targetbiomolecules or which bind the same extracellular target biomolecule attwo or more different epitopes, whether non-overlapping or overlappingepitopes (e.g. a bivalent biparatopic molecule).

As used herein, the terms “expressed,” “expressing,” or “expresses,” andgrammatical variants thereof, refer to translation of a polynucleotideor nucleic acid into a protein. The expressed protein may remainintracellular, become a component of the cell surface membrane or besecreted into an extracellular space.

As used herein, cells which express a significant amount of anextracellular target biomolecule at least one cellular surface are“target positive cells” or “target+cells” and are cells physicallycoupled to the specified, extracellular target biomolecule. Asignificant amount of target biomolecule is defined below.

As used herein, the symbol “a” is shorthand for an immunoglobulin-typebinding region capable of binding to the biomolecule following thesymbol. The symbol “α” is used to refer to the functional characteristicof an immunoglobulin-type binding region based on its ability to bind tothe biomolecule following the symbol with a binding affinity describedby a dissociation constant (K_(D)) of 10⁻⁵ or less.

As used herein, the term “heavy chain variable (V_(H)) domain” or “lightchain variable (V_(L)) domain” respectively refer to any antibody V_(H)or V_(L) domain (e.g. a human V_(H) or V_(L) domain) as well as anyderivative thereof retaining at least qualitative antigen bindingability of the corresponding native antibody (e.g. a humanized V_(H) orV_(L) domain derived from a native murine V_(H) or V_(L) domain). AV_(H) or V_(L) domain consists of a “framework” region interrupted bythe three CDRs or ABRs. The framework regions serve to align the CDRs orABRs for specific binding to an epitope of an antigen. From aminoterminus to carboxy terminus, both V_(H) and V_(L) domains comprise thefollowing framework (FR) and CDR regions or ABR regions; FR1, CDR1, FR2,CDR2, FR3, CDR3, and FR4; or, similarly, FR1, ABR1, FR2, ABR2, FR3,ABR3, and FR4. As used herein, the terms “HCDR1,” “HCDR2,” or “HCDR3”are used to refer to CDRs 1, 2, or 3, respectively, in a V_(H) domain,and the terms “LCDR1,” “LCDR2,” and “LCDR3” are used to refer to CDRs 1,2, or 3, respectively, in a V_(L) domain. As used herein, the terms“HABR1,” “HABR2,” or “HABR3” are used to refer to ABRs 1, 2, or 3,respectively, in a V_(H) domain, and the terms “LABR1,” “LABR2,” or“LABR3” are used to refer to CDRs 1, 2, or 3, respectively, in a V_(L)domain. For camelid V_(H)H fragments, IgNARs of cartilaginous fish.V_(NAR) fragments, certain single domain antibodies, and derivativesthereof, there is a single, heavy chain variable domain comprising thesame basic arrangement: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Asused herein, the terms “HCDR1,” “HCDR2,” or “HCDR3” may be used to referto CDRs 1, 2, or 3, respectively, in a single heavy chain variabledomain.

For purposes of the present invention, the term “effector” meansproviding a biological activity, such as cytotoxicity, biologicalsignaling, enzymatic catalysis, subcellular routing, and/orintermolecular binding resulting in an allosteric effect(s) and/or therecruitment of one or more factors. For example, a Shiga toxin effectorpolypeptide provides one or more biological activities present in aShiga toxin, Shiga toxin component, and/or fragment thereof.

For purposes of the present invention, the phrases “Shiga toxin effectorpolypeptide,” “Shiga toxin effector polypeptide region,” and “Shigatoxin effector region” refer to a polypeptide or polypeptide regionderived from at least one Shiga toxin A Subunit of a member of the Shigatoxin family wherein the polypeptide or polypeptide region is capable ofexhibiting at least one Shiga toxin function.

For purposes of the present invention, the term “heterologous” asdescribing a binding region means the binding region is from a differentsource than a naturally occurring Shiga toxin, e.g. a heterologousbinding region which is a polypeptide is polypeptide not naturally foundas part of any native Shiga toxin.

For purposes of the present invention, the term “heterologous” asdescribing a CD8+ T-cell epitope means the CD8+ T-cell epitope is from adifferent source than (1) an A Subunit of a naturally occurring Shigatoxin, e.g. a heterologous polypeptide is not naturally found as part ofany A Subunit of a native Shiga toxin and (2) a prior art Shiga toxineffector polypeptide. For example, in certain embodiments of thecell-targeting molecules of the present invention, the term“heterologous” with regard to a CD8+ T-cell epitope-peptide refers to apeptide sequence which did not initially occur in a cell-targetingmolecule to be modified (parental molecule), but which was added to themolecule, whether added via the processes of embedding, fusion,insertion, and/or amino acid substitution as described herein, or by anyother engineering means to create a modified cell-targeting molecule.The result is a modified cell-targeting molecule comprising a CD8+T-cell epitope-peptide which is foreign to the original, unmodifiedcell-targeting molecule, i.e. the CD8+ T-cell epitope was not present inthe unmodified cell-targeting molecule (parental molecule). The‘heterologous’ CD8+ T-cell epitope may be autogenous or heterologouswith respect to the binding region.

In certain embodiments of the cell-targeting molecule of the presentinvention, the heterologous, CD8+ T-cell epitope is also heterologous tothe binding region component(s) of the cell-targeting molecule, e.g. aheterologous epitope is one that is not required for the bindingactivity of the binding region and is not part of the structure of theminimum binding region structure which provides the binding activity ofthe binding region. For example, a CD8+ T-cell epitope not nativelypresent in an immunoglobulin is heterologous to an immunoglobulin-typebinding region derived from that immunoglobulin if it is not requiredfor the binding activity of the immunoglobulin-type binding region andis not part of the structure of the minimum binding region structurewhich provides the binding activity of the immunoglobulin-type bindingregion.

For purposes of the claimed invention, the term “heterologous” asdescribing a CD8+ T-cell epitope means of a different source than (1) anA Subunit of a naturally occurring Shiga toxin and (2) the bindingregion of the cell-targeting molecule comprising the heterologouscomponent. A heterologous, CD8+ T-cell epitope-peptide of thecell-targeting molecule of the present invention is an CD8+ T-cellepitope-peptide not already present in a wild-type Shiga toxin A1fragment; a naturally occurring Shiga toxin A1 fragment: and/or a priorart Shiga toxin effector polypeptide used as a component of thecell-targeting molecule.

For purposes of the claimed invention, the phrase “intercellularengagement” by a CD8+ immune cell refers to a CD8+ immune cellresponding to different cell (for example, by sensing the other isdisplaying one or more pMHC Is) in fashion indicative of the activationof an immune response by the CD8+ immune cell, such as, e.g., responsesinvolved in killing the other cell, recruiting and activating otherimmune cells (e.g. cytokine secretion), maturation of the CD8+ immunecell, activation of the CD8+ immune cell, etc.

As used herein, the term “CD8+ T-cell epitope delivering” whendescribing a functional activity of a molecule means that a moleculeprovides the biological activity of localizing within a cell to asubcellular compartment that is competent to result in the proteasomalcleavage of a proteinaceous part of the molecule which comprises a CD8+T-cell epitope-peptide. The “CD8+ T-cell epitope delivering” function ofa molecule can be assayed by observing the MHC presentation of a CD8+T-cell epitope-peptide cargo of the molecule on a cell surface of a cellexogenously administered the molecule or in which the assay was begunwith the cell containing the molecule in one or more of its endosomalcompartments.

Generally, the ability of a molecule to deliver a CD8+ T-cell epitope toa proteasome can be determined where the initial location of the “CD8+T-cell epitope delivering” molecule is an early endosomal compartment ofa cell, and then, the molecule is empirically shown to deliver theepitope-peptide to the proteasome of the cell. However, a “CD8+ T-cellepitope delivering” ability may also be determined where the moleculestarts at an extracellular location and is empirically shown, eitherdirectly or indirectly, to deliver the epitope into a cell and toproteasomes of the cell. For example, certain “CD8+ T-cell epitopedelivering” molecules pass through an endosomal compartment of the cell,such as, e.g. after endocytotic entry into that cell. Alternatively,“CD8+ T-cell epitope delivering” activity may be observed for a moleculestarting at an extracellular location whereby the molecule does notenter any endosomal compartment of a cell-instead the “CD8+ T-cellepitope delivering” molecule enters a cell and delivers a T-cellepitope-peptide to proteasomes of the cell, presumably because the “CD8+T-cell epitope delivering” molecule directed its own routing to asubcellular compartment competent to result in proteasomal cleavage ofits CD8+ T-cell epitope-peptide component.

For purposes of the present invention, a Shiga toxin effector functionis a biological activity conferred by a polypeptide region derived froma Shiga toxin A Subunit. Non-limiting examples of Shiga toxin effectorfunctions include promoting cell entry; lipid membrane deformation;promoting cellular internalization; stimulating clathrin-mediatedendocytosis; directing intracellular routing to various intracellularcompartments such as, e.g., the Golgi, endoplasmic reticulum, andcytosol; directing intracellular routing with a cargo; inhibiting aribosome function(s); catalytic activities, such as, e.g., N-glycosidaseactivity and catalytically inhibiting ribosomes; reducing proteinsynthesis, inducing caspase activation, activating effector caspases,effectuating cytostatic effects, and cytotoxicity. Shiga toxin catalyticactivities include, for example, ribosome inactivation, proteinsynthesis inhibition, N-glycosidase activity, polynucleotide:adenosineglycosidase activity, RNAase activity, and DNAase activity. Shiga toxinsare ribosome inactivating proteins (RIPs). RIPs can depurinate nucleicacids, polynucleosides, polynucleotides, rRNA, ssDNA, dsDNA, mRNA (andpolyA), and viral nucleic acids (see e.g., Barbieri L et al., Biochem J286: 1-4 (1992); Barbieri L et al., Nature 372: 624 (1994); Ling J etal., FEBS Lett 345: 143-6 (1994); Barbieri L et al., Biochem J 319:507-13 (1996); Roncuzzi L, Gasperi-Campani A, FEBS Lett 392: 16-20(1996); Stirpe F et al., FEBSLett 382: 309-12 (1996); Barbieri L et al.,Nucleic Acids Res 25: 518-22 (1997); Wang P, Turner N, Nucleic Acids Res27: 1900-5 (1999); Barbieri L et al., Biochim Biophys Acta 1480: 258-66(2000); Barbieri L et al., J Biochem 128: 883-9 (2000); Brigotti M etal., Toxicon 39: 341-8 (2001); Brigotti M et al., FASEB J 16: 365-72(2002); Bagga S et al., J Biol Chem 278: 4813-20 (2003); Picard D et al.J Biol Chem 280: 20069-75 (2005)). Some RIPs show antiviral activity andsuperoxide dismutase activity (Erice A et al., Antimicrob AgentsChemother 37: 835-8 (1993); Au T et al., FEBS Lett 471: 169-72 (2000);Parikh B. Turner N, Mini Rev Med Chem 4: 523-43 (2004); Sharma N et al.,Plant Physiol 134: 171-81 (2004)). Shiga toxin catalytic activities havebeen observed both in vitro and in vivo. Non-limiting examples of assaysfor Shiga toxin effector activity measure various activities, such as,e.g., protein synthesis inhibitory activity, depurination activity,inhibition of cell growth, cytotoxicity, supercoiled DNA relaxationactivity, and nuclease activity.

As used herein, the retention of Shiga toxin effector function refers tobeing capable of exhibiting a level of Shiga toxin functional activity,as measured by an appropriate quantitative assay with reproducibility,comparable to a wild-type, Shiga toxin effector polypeptide control(e.g. a Shiga toxin A1 fragment) or cell-targeting molecule comprising awild-type Shiga toxin effector polypeptide (e.g. a Shiga toxin A1fragment) under the same conditions. For the Shiga toxin effectorfunction of ribosome inactivation or ribosome inhibition, retained Shigatoxin effector function is exhibiting an IC₅₀ of 10,000 picomolar (pM)or less in an in vitro setting, such as, e.g., by using an assay knownto the skilled worker and/or described herein. For the Shiga toxineffector function of cytotoxicity in a target positive cell-kill assay,retained Shiga toxin effector function is exhibiting a CD₅₀ of 1,000nanomolar (nM) or less, depending on the cell-type and its expression ofthe appropriate extracellular target biomolecule, as shown, e.g., byusing an assay known to the skilled worker and/or described herein.

For purposes of the claimed invention, the term “equivalent” with regardto ribosome inhibition means an empirically measured level of ribosomeinhibitory activity, as measured by an appropriate quantitative assaywith reproducibility, which is reproducibly within 10% or less of theactivity of the reference molecule (e.g., the second cell-targetingmolecule or third cell-targeting molecule) under the same conditions.

For purposes of the claimed invention, the term “equivalent” with regardto cytotoxicity means an empirically measured level of cytotoxicity, asmeasured by an appropriate quantitative assay with reproducibility,which is reproducibly within 10% or less of the activity of thereference molecule (e.g., the second cell-targeting molecule or thirdcell-targeting molecule) under the same conditions.

As used herein, the term “attenuated” with regard to cytotoxicity meansa molecule exhibits or exhibited a CD₅₀ between 10-fold to 100-fold of aCD₅₀ exhibited by a reference molecule under the same conditions.

For some samples, accurate values for either IC₅₀ or CD₅₀ might beunobtainable due to the inability to collect the required data pointsfor an accurate curve fit. For example, theoretically, neither an IC₅₀nor CD₅₀ can be determined if greater than 50% ribosome inhibition orcell death, respectively, does not occur in a concentration series for agiven sample. Data insufficient to accurately fit a curve as describedin the analysis of the data from exemplary Shiga toxin effector functionassays, such as, e.g., assays described in the Examples, should not beconsidered as representative of actual Shiga toxin effector function.

A failure to detect activity in Shiga toxin effector function may be dueto improper expression, polypeptide folding, and/or polypeptidestability rather than a lack of cell entry, subcellular routing, and/orenzymatic activity. Assays for Shiga toxin effector functions may notrequire much cell-targeting molecule of the invention to measuresignificant amounts of Shiga toxin effector function activity.

To the extent that an underlying cause of low or no effector function isdetermined empirically to relate to protein expression or stability, oneof skill in the art may be able to compensate for such factors usingprotein chemistry and molecular engineering techniques known in the art,such that a Shiga toxin functional effector activity may be restored andmeasured. As examples, improper cell-based expression may be compensatedfor by using different expression control sequences; improperpolypeptide folding and/or stability may benefit from stabilizingterminal sequences, or compensatory mutations in non-effector regionswhich stabilize the three-dimensional structure of the protein, etc.When new assays for individual Shiga toxin functions become available,Shiga toxin effector regions or polypeptides may be analyzed for anylevel of those Shiga toxin effector functions, such as for being withina certain-fold activity of a wild-type Shiga toxin effector polypeptide.Examples of meaningful activity differences are, e.g., Shiga toxineffector regions that have 1000-fold or 100-fold or less the activity ofa wild-type Shiga toxin effector polypeptide; or that have 3-fold to30-fold or more activity compared to a functional knock-down or knockoutShiga toxin effector polypeptide.

Certain Shiga toxin effector functions are not easily measurable, e.g.subcellular routing functions. For example, there is no routine,quantitative assay to distinguish whether the failure of a Shiga toxineffector polypeptide to be cytotoxic and/or deliver a heterologous, CD8+T-cell epitope is due to improper subcellular routing, but at a timewhen tests are available, then Shiga toxin effector polypeptides may beanalyzed for any significant level of subcellular routing as compared tothe appropriate wild-type Shiga toxin effector polypeptide. However, ifa Shiga toxin effector polypeptide component of a cell-targetingmolecule of the present invention exhibits cytotoxicity comparable orequivalent to a wild-type Shiga toxin A Subunit construct, then thesubcellular routing activity level is inferred to be comparable orequivalent, respectively, to the subcellular routing activity level of awild-type Shiga toxin A Subunit construct at least under the conditionstested.

When new assays for individual Shiga toxin functions become available,Shiga toxin effector polypeptides and/or cell-targeting moleculescomprising Shiga toxin effector polypeptides may be analyzed for anylevel of those Shiga toxin effector functions, such as a being within1000-fold or 100-fold or less the activity of a wild-type Shiga toxineffector polypeptide or exhibiting 3-fold to 30-fold or greater activityas compared to a functional knockout, Shiga toxin effector polypeptide.

Sufficient subcellular routing may be merely deduced by observing acell-targeting molecule's Shiga toxin cytotoxic activity levels incytotoxicity assays, such as, e.g., cytotoxicity assays based on T-cellepitope presentation or based on a Shiga toxin effector functioninvolving a cytosolic and/or endoplasmic reticulum-localized, targetsubstrate.

As used herein, the retention of “significant” Shiga toxin effectorfunction refers being capable of exhibiting a level of Shiga toxinfunctional activity, as measured by an appropriate quantitative assaywith reproducibility comparable to a wild-type Shiga toxin effectorpolypeptide control (e.g. a Shiga toxin A1 fragment). For in vitroribosome inhibition, significant Shiga toxin effector function isexhibiting an IC₅₀ of 300 pM or less depending on the source of theribosomes used in the assay (e.g. a bacterial, archaeal, or eukaryotic(algal, fungal, plant, or animal) source). This is significantly greaterinhibition as compared to the approximate IC₅₀ of 100,000 pM for thecatalytically disrupted SLT-1A 1-251 double mutant (Y77S/E167D). Forcytotoxicity in a target-positive cell-kill assay in laboratory cellculture, significant Shiga toxin effector function is exhibiting a CD₅₀of 100, 50, 30 nM, or less, depending on the target biomolecule(s) ofthe binding region and the cell-type, particularly that cell-type'sexpression and/or cell-surface representation of the appropriateextracellular target biomolecule(s) and/or the extracellular epitope(s)targeted by the molecule being evaluated. This is significantly greatercytotoxicity to the appropriate, target-positive cell population ascompared to a Shiga toxin A Subunit alone (or a wild-type Shiga toxin A1fragment), without a cell targeting binding region, which has a CD₅₀ of100-10,000 nM, depending on the cell line.

For purposes of the present invention and with regard to the Shiga toxineffector function of a molecule of the present invention, the term“reasonable activity” refers to exhibiting at least a moderate level(e.g. within 1-fold to 1,000-fold) of Shiga toxin effector activity asdefined herein in relation to a molecule comprising a naturallyoccurring Shiga toxin, wherein the Shiga toxin effector activity isselected from the group consisting of: internalization efficiency,subcellular routing efficiency to the cytosol, delivered epitopepresentation by a target cell(s), ribosome inhibition, and cytotoxicity.For cytotoxicity, a reasonable level of Shiga toxin effector activityincludes being within 1,000-fold of a wild-type, Shiga toxin construct,such as, e.g., exhibiting a CD₅₀ of 500 nM or less when a wild-typeShiga toxin construct exhibits a CD₅₀ of 0.5 nM (e.g. a cell-targetingmolecule comprising a wild-type Shiga toxin A1 fragment).

For purposes of the present invention and with regard to thecytotoxicity of a molecule of the present invention, the term “optimal”refers to a level of Shiga toxin catalytic domain mediated cytotoxicitythat is within 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold of the cytotoxicity ofa molecule comprising wild-type Shiga toxin A1 fragment (e.g. a Shigatoxin A Subunit or certain truncated variants thereof) and/or anaturally occurring Shiga toxin.

It should be noted that even if the cytotoxicity of a Shiga toxineffector polypeptide is reduced relative to a wild-type Shiga toxin ASubunit or fragment thereof, in practice, applications using biologicalactivity-attenuated, Shiga toxin effector polypeptides may be equally ormore effective than using wild-type Shiga toxin effector polypeptidesbecause the highest potency variants might exhibit undesirable effectswhich are minimized or reduced in reduced cytotoxic-potency variants.Wild-type Shiga toxins are very potent, being able to kill anintoxicated cell after only one toxin molecule has reached the cytosolof the intoxicated cell or perhaps after only forty toxin molecules havebeen internalized into the intoxicated cell. Shiga toxin effectorpolypeptides with even considerably reduced Shiga toxin effectorfunctions, such as, e.g., subcellular routing or cytotoxicity, ascompared to wild-type Shiga toxin effector polypeptides may still bepotent enough for practical applications, such as, e.g., applicationsinvolving targeted cell-killing, heterologous epitope delivery, and/ordetection of specific cells and their subcellular compartments. Inaddition, certain reduced-activity Shiga toxin effector polypeptides maybe particularly useful for delivering cargos (e.g. an additionalexogenous material or T-cell epitope) to certain intracellular locationsor subcellular compartments of target cells.

The term “selective cytotoxicity” with regard to the cytotoxic activityof a molecule refers to the relative level of cytotoxicity between abiomolecule target positive cell population (e.g. a targeted cell-type)and a non-targeted bystander cell population (e.g. a biomolecule targetnegative cell-type), which can be expressed as a ratio of thehalf-maximal cytotoxic concentration (CD₅₀) for a targeted cell-typeover the CD₅₀ for an untargeted cell-type to provide a metric ofcytotoxic selectivity or indication of the preferentiality of killing ofa targeted cell versus an untargeted cell.

The cell surface representation and/or density of a given extracellulartarget biomolecule (or extracellular epitope of a given targetbiomolecule) may influence the applications for which certaincell-targeting molecules of the present invention may be most suitablyused. Differences in cell surface representation and/or density of agiven target biomolecule between cells may alter, both quantitativelyand qualitatively, the efficiency of cellular internalization and/orcytotoxicity potency of a given cell-targeting molecule of the presentinvention. The cell surface representation and/or density of a giventarget biomolecule can vary greatly among target biomolecule positivecells or even on the same cell at different points in the cell cycle orcell differentiation. The total cell surface representation of a giventarget biomolecule and/or of certain extracellular epitopes of a giventarget biomolecule on a particular cell or population of cells may bedetermined using methods known to the skilled worker, such as methodsinvolving fluorescence-activated cell sorting (FACS) flow cytometry.

For purposes of the present invention, the phrase “target biomoleculenatively present on the surface of a cell” means a cell expresses thetarget biomolecule using its own internal machinery and localizes thetarget biomolecule to a cellular surface using its own internalmachinery such that the target biomolecule is physically coupled to saidcell and at least a part of the target biomolecule is accessible from anextracellular space, i.e. on the surface of a cell.

As used herein, the terms “disrupted,” “disruption,” or “disrupting,”and grammatical variants thereof, with regard to a polypeptide region orfeature within a polypeptide refers to an alteration of at least oneamino acid within the region or composing the disrupted feature. Aminoacid alterations include various mutations, such as, e.g., a deletion,inversion, insertion, or substitution which alter the amino acidsequence of the polypeptide. Amino acid alterations also includechemical changes, such as, e.g., the alteration one or more atoms in anamino acid functional group or the addition of one or more atoms to anamino acid functional group.

As used herein. “de-immunized” means reduced antigenic and/orimmunogenic potential after administration to a chordate as compared toa reference molecule, such as, e.g., a wild-type peptide region,polypeptide region, or polypeptide. This includes a reduction in overallantigenic and/or immunogenic potential despite the introduction of oneor more, de novo, antigenic and/or immunogenic epitopes as compared to areference molecule. For certain embodiments, “de-immunized” means amolecule exhibited reduced antigenicity and/or immunogenicity afteradministration to a mammal as compared to a “parental” molecule fromwhich it was derived, such as, e.g., a wild-type Shiga toxin A1fragment. In certain embodiments, the de-immunized, Shiga toxin effectorpolypeptide of the present invention is capable of exhibiting a relativeantigenicity compared to a reference molecule which is reduced by 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater than the antigenicityof the reference molecule under the same conditions measured by the sameassay, such as, e.g., an assay known to the skilled worker and/ordescribed herein like a quantitative ELISA or Western blot analysis. Incertain embodiments, the de-immunized, Shiga toxin effector polypeptideof the present invention is capable of exhibiting a relativeimmunogenicity compared to a reference molecule which is reduced by 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or greater thanthe immunogenicity of the reference molecule under the same conditionsmeasured by the same assay, such as, e.g., an assay known to the skilledworker and/or described herein like a quantitative measurement ofanti-molecule antibodies produced in a mammal(s) after receivingparenteral administration of the molecule at a given time-point. Therelative immunogenicities of exemplary cell-targeting molecules may bedetermined using an assay for adaptive immune reactions (e.g. in vivoantibody responses) or innate immune reactions to the cell-targetingmolecules after repeat, parenteral administrations over periods of days,weeks, and/or months.

The relative immunogenicities of exemplary cell-targeting molecules weredetermined using an assay for in vivo antibody responses to thecell-targeting molecules after repeat, parenteral administrations overperiods of many.

For purposes of the present invention, the phrase “CD8+ T-cellhyper-immunized” means that the cell-targeting molecule, when presentinside a nucleated, chordate cell within a living chordate, has anincreased antigenic and/or immunogenic potential regarding CD8+ T-cellantigenicity or immunogenicity when compared to the same molecule thatlacks any heterologous, CD8+ T-cell epitope-peptide.

The term “embedded” and grammatical variants thereof with regard to aT-cell epitope or T-cell epitope-peptide component of a polypeptiderefers to the internal replacement of one or more amino acids within apolypeptide region with different amino acids in order to generate a newpolypeptide sequence sharing the same total number of amino acidresidues with the starting polypeptide region.

Thus, the term “embedded” does not include any external, terminal fusionof any additional amino acid, peptide, or polypeptide component to thestarting polypeptide nor any additional internal insertion of anyadditional amino acid residues, but rather includes only substitutionsfor existing amino acids. The internal replacement may be accomplishedmerely by amino acid residue substitution or by a series ofsubstitutions, deletions, insertions, and/or inversions. If an insertionof one or more amino acids is used, then the equivalent number ofproximal amino acids must be deleted next to the insertion to result inan embedded T-cell epitope. This is in contrast to use of the term“inserted” with regard to a T-cell epitope contained within apolypeptide of the present invention to refer to the insertion of one ormore amino acids internally within a polypeptide resulting in a newpolypeptide having an increased number of amino acids residues comparedto the starting polypeptide.

The term “inserted” and grammatical variants thereof with regard to aT-cell epitope contained within a polypeptide refers to the insertion ofone or more amino acids within a polypeptide resulting in a newpolypeptide sequence having an increased number of amino acids residuescompared to the starting polypeptide.

For purposes of the present invention, the phrase “proximal to an aminoterminus” with reference to the position of a Shiga toxin effectorpolypeptide region of a cell-targeting molecule of the present inventionrefers to a distance wherein at least one amino acid residue of theShiga toxin effector polypeptide region is within 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12 or more, e.g., up to 18-20 amino acid residues, of anamino terminus of the cell-targeting molecule as long as thecell-targeting molecule is capable of exhibiting the appropriate levelof Shiga toxin effector functional activity noted herein (e.g., acertain level of cytotoxic potency). Thus for certain embodiments of thepresent invention, any amino acid residue(s) fused amino-terminal to theShiga toxin effector polypeptide should not reduce any Shiga toxineffector function (e.g., by sterically hindering a structure(s) near theamino terminus of the Shiga toxin effector polypeptide region) such thata functional activity of the Shiga toxin effector polypeptide is reducedbelow the appropriate activity level required herein.

For purposes of the present invention, the phrase “more proximal to anamino terminus” with reference to the position of a Shiga toxin effectorpolypeptide region within a cell-targeting molecule of the presentinvention as compared to another component (e.g., a cell-targeting,binding region, molecular moiety, and/or additional exogenous material)refers to a position wherein at least one amino acid residue of theamino terminus of the Shiga toxin effector polypeptide is closer to theamino terminus of a linear, polypeptide component of the cell-targetingmolecule of the present invention as compared to the other referencedcomponent.

For purposes of the present invention, the phrase “active enzymaticdomain derived from one A Subunit of a member of the Shiga toxin family”refers to having the ability to inhibit protein synthesis via acatalytic ribosome inactivation mechanism. The enzymatic activities ofnaturally occurring Shiga toxins may be defined by the ability toinhibit protein translation using assays known to the skilled worker,such as, e.g., in vitro assays involving RNA translation in the absenceof living cells or in vivo assays involving RNA translation in a livingcell. Using assays known to the skilled worker and/or described herein,the potency of a Shiga toxin enzymatic activity may be assessed directlyby observing N-glycosidase activity toward ribosomal RNA (rRNA), suchas, e.g., a ribosome nicking assay, and/or indirectly by observinginhibition of ribosome function and/or protein synthesis.

For purposes of the present invention, the term “Shiga toxin A1 fragmentregion” refers to a polypeptide region consisting essentially of a Shigatoxin A1 fragment and/or derived from a Shiga toxin A1 fragment of aShiga toxin.

For purposes of the present invention, the terms “terminus,” “aminoterminus,” or “carboxy terminus” with regard to a cell-targetingmolecule refers generally to the last amino acid residue of apolypeptide chain of the cell-targeting molecule (e.g., a single,continuous polypeptide chain). A cell-targeting molecule may comprisemore than one polypeptide or protein, and, thus, a cell-targetingmolecule of the present invention may comprise multiple amino-terminalsand carboxy-terminals. For example, the “amino terminus” of acell-targeting molecule may be defined by the first amino acid residueofa polypeptide chain representing the amino-terminal end of thepolypeptide, which is generally characterized by a starting, amino acidresidue which does not have a peptide bond with any amino acid residueinvolving the primary amino group of the starting amino acid residue orinvolving the equivalent nitrogen for starting amino acid residues whichare members of the class of N-alkylated alpha amino acid residues.Similarly, the “carboxy terminus” of a cell-targeting molecule may bedefined by the last amino acid residue of a polypeptide chainrepresenting the carboxyl-terminal end of the polypeptide, which isgenerally characterized by a final, amino acid residue which does nothave any amino acid residue linked by a peptide bond to the alpha-carbonof its primary carboxyl group.

For purposes of the present invention, the phrase “Shiga toxin A1fragment derived region” refers to all or part of a Shiga toxin effectorpolypeptide wherein the region consists of a polypeptide homologous to anaturally occurring Shiga toxin A1 fragment or truncation thereof, suchas, e.g., a polypeptide consisting of or comprising amino acids 75-239of SLT-1A (SEQ ID NO: 1), 75-239 of StxA (SEQ ID NO:2), or 77-238 of(SEQ ID NO:3) or the equivalent region in another A Subunit of a memberof the Shiga toxin family. The carboxy-terminus of a “Shiga toxin A1fragment derived region” is defined, relative to a naturally occurringShiga toxin A1 fragment, (1) as ending with the carboxy-terminal aminoacid residue sharing homology with a naturally occurring, Shiga toxin A1fragment; (2) as ending at the junction of the A1 fragment and the A2fragment; (3) as ending with a furin-cleavage site or disruptedfurin-cleave site; and/or (4) as ending with a carboxy-terminaltruncation of a Shiga toxin A1 fragment, i.e. the carboxy-terminal aminoacid residue sharing homology with a naturally occurring, Shiga toxin A1fragment.

For purposes of the present invention, the phrase “carboxy terminusregion of a Shiga toxin A1 fragment” refers to a polypeptide regionderived from a naturally occurring Shiga toxin A1 fragment, the regionbeginning with a hydrophobic residue (e.g., V236 of StxA-A1 and SLT-1A1,and V235 of SLT-2A1) that is followed by a hydrophobic residue and theregion ending with the furin-cleavage site conserved among Shiga toxinA1 fragment polypeptides and ending at the junction between the A1fragment and the A2 fragment in native, Shiga toxin A Subunits. Forpurposes of the present invention, the carboxy-terminal region of aShiga toxin A1 fragment includes a peptidic region derived from thecarboxy terminus of a Shiga toxin A1 fragment polypeptide, such as,e.g., a peptidic region comprising or consisting essentially of thecarboxy terminus of a Shiga toxin A1 fragment. Non-limiting examples ofpeptidic regions derived from the carboxy terminus of a Shiga toxin A1fragment include the amino acid residue sequences natively positionedfrom position 236 to position 239, 240, 241, 242, 243, 244, 245, 246,247, 248, 249, 250, or 251 in Shiga toxin A subunit variants (SEQ IDNOs: 1-2 and 4-6); and from position 235 to position 239, 240, 241, 242,243, 244, 245, 246, 247, 248, 249, or 250 in SLT-2A variants (SEQ IDNOs: 3 and 7-18).

For purposes of the present invention, the phrase “proximal to thecarboxy terminus of an A1 fragment polypeptide” with regard to a linkedmolecular moiety and/or binding region refers to being within 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues from the amino acidresidue defining the last residue of the Shiga toxin A1 fragmentpolypeptide.

For purposes of the present invention, the phrase “sterically covers thecarboxy terminus of the A1 fragment-derived region” includes anymolecular moiety of a size of 4.5 kDa or greater (e.g., animmunoglobulin-type binding region) linked and/or fused to an amino acidresidue in the carboxy terminus of the A1 fragment-derived region, suchas, e.g., the amino acid residue derived from the amino acid residuenatively positioned at any one of positions 236 to 251 in Shiga toxinvariants (SEQ ID NOs: 1-2 and 4-6) or from 235 to 250 in SLT-2A variants(SEQ ID NOs: 3 and 7-18). For purposes of the present invention, thephrase “sterically covers the carboxy terminus of the A1fragment-derived region” also includes any molecular moiety of a size of4.5 kDa or greater (e.g., an immunoglobulin-type binding region) linkedand/or fused to an amino acid residue in the carboxy terminus of the A1fragment-derived region, such as, e.g., the amino acid residuecarboxy-terminal to the last amino acid A1 fragment-derived regionand/or the Shiga toxin effector polypeptide. For purposes of the presentinvention, the phrase “sterically covers the carboxy terminus of the A1fragment-derived region” also includes any molecular moiety of a size of4.5 kDa or greater (e.g., an immunoglobulin-type binding region)physically preventing cellular recognition of the carboxy terminus ofthe A1 fragment-derived region, such as, e.g. recognition by the ERADmachinery of a eukaryotic cell.

For purposes of the present invention, a binding region, such as, e.g.,an immunoglobulin-type binding region, that comprises a polypeptidecomprising at least forty amino acids and that is linked (e.g., fused)to the carboxy terminus of the Shiga toxin effector polypeptide regioncomprising an A1 fragment-derived region is a molecular moiety which is“sterically covering the carboxy terminus of the A1 fragment-derivedregion.”

For purposes of the present invention, a binding region, such as, e.g.,an immunoglobulin-type binding region, that comprises a polypeptidecomprising at least forty amino acids and that is linked (e.g., fused)to the carboxy terminus of the Shiga toxin effector polypeptide regioncomprising an A1 fragment-derived region is a molecular moiety“encumbering the carboxy terminus of the A1 fragment-derived region.”

For purposes of the present invention, the term “A fragment of a memberof the Shiga toxin family” refers to the remaining amino-terminalfragment of a Shiga toxin A Subunit after proteolysis by furin at thefurin-cleavage site conserved among Shiga toxin A Subunits andpositioned between the A1 fragment and the A2 fragment in wild-typeShiga toxin A Subunits.

For purposes of the claimed invention, the phrase “furin-cleavage motifat the carboxy terminus of the A1 fragment region” refers to a specific,furin-cleavage motif conserved among Shiga toxin A Subunits and bridgingthe junction between the A1 fragment and the A2 fragment in naturallyoccurring, Shiga toxin A Subunits.

For purposes of the present invention, the phrase “furin-cleavage siteproximal to the carboxy terminus of the A1 fragment region” refers toany identifiable, furin-cleavage site having an amino acid residuewithin a distance of less than 1, 2, 3, 4, 5, 6, 7, or more amino acidresidues of the amino acid residue defining the last amino acid residuein the A1 fragment region or A1 fragment derived region, including afurin-cleavage motif located carboxy-terminal of an A1 fragment regionor A1 fragment derived region, such as, e.g., at a position proximal tothe linkage of the A1 fragment-derived region to another component ofthe molecule, such as, e.g., a molecular moiety of a cell-targetingmolecule of the present invention.

For purposes of the present invention, the phrase “disruptedfurin-cleavage motif” refers to (i) a specific furin-cleavage motif asdescribed herein and (ii) which comprises a mutation and/or truncationthat can confer a molecule with a reduction in furin-cleavage ascompared to a reference molecule, such as, e.g., a reduction infurin-cleavage reproducibly observed to be 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 97%, 98%, 99%, or less (including 100% for no cleavage) thanthe furin-cleavage of a reference molecule observed in the same assayunder the same conditions. The percentage of furin-cleavage as comparedto a reference molecule can be expressed as a ratio of cleaved/uncleavedmaterial of the molecule of interest divided by the cleaved/uncleavedmaterial of the reference molecule (see e.g. WO 2015/191764; US2016/0177284). Non-limiting examples of suitable reference moleculesinclude certain molecules comprising a wild-type Shiga toxinfurin-cleavage motif and/or furin-cleavage site as described hereinand/or molecules used in the Examples below.

For purposes of the present invention, the phrase “furin-cleavageresistant” means a molecule or specific polypeptide region thereofexhibits reproducibly less furin cleavage than (i) the carboxy terminusof a Shiga toxin A1 fragment in a wild-type Shiga toxin A Subunit or(ii) the carboxy terminus of the Shiga toxin A1 fragment derived regionof construct wherein the naturally occurring furin-cleavage sitenatively positioned at the junction between the A1 and A2 fragments isnot disrupted; as assayed by any available means to the skilled worker,including by using a method described herein.

For purposes of the present invention, the phrase “active enzymaticdomain derived form an A Subunit of a member of the Shiga toxin family”refers to a polypeptide structure having the ability to inhibit proteinsynthesis via catalytic inactivation of a ribosome based on a Shigatoxin enzymatic activity. The ability of a molecular structure toexhibit inhibitory activity of protein synthesis and/or catalyticinactivation of a ribosome may be observed using various assays known tothe skilled worker, such as, e.g., in vitro assays involving RNAtranslation assays in the absence of living cells or in vivo assaysinvolving the ribosomes of living cells. For example, using assays knownto the skilled worker, the enzymatic activity of a molecule based on aShiga toxin enzymatic activity may be assessed directly by observingN-glycosidase activity toward ribosomal RNA (rRNA), such as, e.g., aribosome nicking assay, and/or indirectly by observing inhibition ofribosome function, RNA translation, and/or protein synthesis.

As used herein with respect to a Shiga toxin effector polypeptide, a“combination” describes a Shiga toxin effector polypeptide comprisingtwo or more sub-regions wherein each sub-region comprises at least oneof the following: (1) a disruption in an endogenous epitope or epitoperegion and (2) a disrupted furin-cleavage motif at the carboxy terminusof a Shiga toxin A1 fragment derived region.

As used herein, the term “multivalent” with regard to a cell-targetingmolecule refers to an individual target-binding molecule or plurality oftarget-binding molecules comprising two or more high-affinity bindingregions, such as, e.g. a protein comprising two or more binding regionswherein each individual binding region has a dissociation constant of10⁻⁵ to 10⁻¹² moles per liter toward an extracellular part of a targetbiomolecule.

As used herein, the term “monomeric” with regard to describing a proteinand/or proteinaceous molecule refers to a molecule comprising only onepolypeptide component consisting of a single, continuous polypeptide,regardless of its secondary or tertiary structure, which may besynthesized by a ribosome from a single polynucleotide template,including a continuous linear polypeptide which later forms a cyclicstructure. In contrast, a multimeric molecule comprises two or morepolypeptides (e.g. subunits) which together do not form a single,continuous polypeptide that may be synthesized by a ribosome from asingle polynucleotide template.

As used herein, the term “multimeric” with regard to describing aprotein and/or proteinaceous molecule refers to a molecule thatcomprises two or more, individual, polypeptide components associatedtogether and/or linked together, such as, e.g., a molecule consisting oftwo components each of which is its own continuous polypeptide. Forexample, the association or linkage between components of a molecule mayinclude 1) one or more non-covalent interactions; 2) one or morepost-translational, covalent interactions; 3) one or more, covalentchemical conjugations; and/or 4) one or more covalent interactionsresulting in a single molecule comprising a non-linear polypeptide, suchas, e.g., a branched or cyclic polypeptide structure, resulting from thearrangement of the two or more polypeptide components. A moleculecomprising two, discontinuous polypeptides as a result of theproteolytic cleavage of one or more peptide bonds in a single,continuous polypeptide synthesized by a ribosome from a singlepolynucleotide templates is “multimeric” and not “monomeric.”

The present invention is described more fully hereinafter usingillustrative, non-limiting embodiments, and references to theaccompanying figures. This invention may, however, be embodied in manydifferent forms and should not be construed as to be limited to theembodiments set forth below. Rather, these embodiments are provided sothat this disclosure is thorough and conveys the scope of the inventionto those skilled in the art.

Introduction

It may be possible to harness the power of the immune system by inducinginfection-like immune reactions specifically toward malignant cells(e.g. tumor cells) and/or malignant tissue loci (e.g. tumors) within apatient specifically by using a highly immunogenic, foreign epitope frominfectious agent in order to locally activate a variety of beneficialimmune responses and to specifically mark targeted cells (e.g. tumorcells) as being foreign by inducing an imitation of an infected state.Alternatively, this approach could use highly immunogenic neoepitopes(derived from either infectious or non-infectious agents) or highlyimmunogenic, non-self epitopes derived from non-infectious agents, suchas, e.g., tumor-specific antigens, tumor-associated antigens, andmolecules from plants, fungi, etc.

The present invention exploits the abilities of Shiga toxin A Subunitderived polypeptides to drive their own subcellular routing in order todeliver highly immunogenic, CD8+ T-cell antigens, such as e.g.peptide-epitopes, to the MHC class I presentation system of a chordatecell. The present invention provides various exemplary, Shiga toxin ASubunit derived constructs capable of delivering heterologous, CD8+T-cell epitopes to the MHC class I system of a target cell resulting incell-surface presentation of the delivered epitope wherein the Shigatoxin effector polypeptide components comprise a combination ofmutations providing (1) de-immunization, (2) a reduction in proteasesensitivity, and/or (3) an embedded. T-cell epitope(s). Certainpeptide-epitopes presented in complexes with MHC class I molecules on acellular surface can signal CD8+ effector T-cells to kill the presentingcell as well as stimulate other immune responses in the local area.Thus, the present invention provides Shiga toxin A Subunit derived,cell-targeting molecules which kill specific target cells, such as,e.g., via presentation of certain CD8+ T-cell epitope-peptides by theMHC class I pathway. The cell-targeting molecules of the presentinvention may be utilized, e.g., as cell-killing molecules, cytotoxictherapeutics, therapeutic delivery agents, and diagnostic molecules.

1. The General Structure of the Cell-Targeting Molecules of the PresentInvention

The cell-targeting molecules of the present invention each comprise 1) acell-targeting binding region, 2) a Shiga toxin A Subunit effectorpolypeptide, and 3) a CD8+ T-cell epitope-peptide which is not embeddedor inserted in the Shiga toxin A1 fragment region and which isheterologous to Shiga toxin A Subunits and the binding region of themolecule. This system is modular, in that any number of diverse, CD8+T-cell epitope-peptides may be used as cargos for delivery to the MHCclass I presentation pathway of target cells by the cell-targetingmolecules of the present invention.

A. The General Structures of the Shiga Toxin Effector PolypeptideComponents of the Cell-Targeting Molecules of the Present Invention

The Shiga toxin effector polypeptides and cell-targeting molecules ofthe present invention comprise at least one, Shiga toxin effectorpolypeptide derived from wild-type Shiga toxin A Subunits but compriseone or more structural modifications, such as, e.g., a mutation like atruncation and/or amino acid residue substitution(s). For certainembodiments, the present invention involves the engineering of improved,Shiga toxin A Subunit effector polypeptides comprising the combinationof two or more of the following Shiga toxin effector polypeptidesub-regions; (1) a de-immunized sub-region, (2) a protease-cleavageresistant sub-region near the carboxy-terminus of a Shiga toxin A1fragment region, and (3) a T-cell epitope-peptide embedded or insertedsub-region.

A Shiga toxin effector polypeptide of a cell-targeting molecule of thepresent invention is a polypeptide derived from a Shiga toxin A Subunitmember of the Shiga toxin family that is capable of exhibiting one ormore Shiga toxin functions.

There are numerous Shiga toxin effector polypeptides that are suitablefor use in the present invention or to use as parental polypeptides tobe modified into a Shiga toxin effector polypeptide of the presentinvention using techniques known the art (see e.g., Cheung M et al., MolCancer 9: 28 (2010); WO 2014/164693; WO 2015/113005; WO 2015/113007; WO2015/138452: WO 2015/191764). Shiga toxin functions include, e.g.,promoting cell entry, increasing cellular internalization, directingretrograde transport, directing subcellular routing, directingsubcellular routing from an endosomal compartment to the cytosol,avoiding intracellular degradation, catalytically inactivatingribosomes, effectuating cytotoxicity, and effectuating cytostaticeffects.

The Shiga toxin family of protein toxins is composed of variousnaturally occurring toxins which are structurally and functionallyrelated, e.g., Shiga toxin, Shiga-like toxin I, and Shiga-like toxin 2(Johannes L, R6mer W, Nat Rev Microbiol 8: 105-16 (2010)). Holotoxinmembers of the Shiga toxin family contain targeting domains thatpreferentially bind a specific glycosphingolipid present on the surfaceof some host cells and an enzymatic domain capable of permanentlyinactivating ribosomes once inside a cell (Johannes L, Römer W, Nat RevMicrobiol 8: 105-16 (2010)). Members of the Shiga toxin family share thesame overall structure and mechanism of action (Engedal N et al.,Microbial Biotech 4: 32-46 (2011)). For example, Stx, SLT-1 and SLT-2display indistinguishable enzymatic activity in cell free systems (HeadS et al., J Biol Chem 266: 3617-21 (1991); Tesh V et al., Infect Immun61: 3392-402 (1993); Brigotti M et al., Toxicon 35: 1431-7 (1997)).

The Shiga toxin family encompasses true Shiga toxin (Stx) isolated fromS. dysenteriae serotype 1, Shiga-like toxin 1 variants (SLT1 or Stx1 orSLT-1 or Slt-I) isolated from serotypes of enterohemorrhagic E, coli,and Shiga-like toxin 2 variants (SLT2 or Stx2 or SLT-2) isolated fromserotypes of enterohemorrhagic E. coli. SLT1 differs by only one aminoacid residue from Stx, and both have been referred to as Verocytotoxinsor Verotoxins (VTs) (O'Brien A. Curr Top Microbiol Immunol 180: 65-94(1992)). Although SLT1 and SLT2 variants are only about 53-60% similarto each other at the primary amino acid sequence level, they sharemechanisms of enzymatic activity and cytotoxicity common to the membersof the Shiga toxin family (Johannes L, Römer W, Nat Rev Microbiol 8:105-16 (2010)). Over 39 different Shiga toxins have been described, suchas the defined subtypes Stx1a, Stx1c, Stx1d, Stx1e, Stx2a-g, andStx2dact (Scheutz F et al., J Clin Microbiol 50: 2951-63 (2012); ProbertW et al., J Clin Microbiol 52: 2346-51 (2014)). Members of the Shigatoxin family are not naturally restricted to any bacterial speciesbecause Shiga-toxin-encoding genes can spread among bacterial speciesvia horizontal gene transfer (Strauch E et al., Infect Immun 69: 7588-95(2001); Bielaszewska M et al., Appl Environ Micrbiol 73: 3144-50 (2007);Zhaxybayeva O, Doolittle W, Curr Biol 21: R242-6 (2011); Kruger A,Lucchesi P, Microbiology 161: 451-62 (2015)). As an example ofinterspecies transfer, a Shiga toxin was discovered in a strain of A.haemolyticus isolated from a patient (Grotiuz G et al., J Clin Microbiol44: 3838-41 (2006)). Once a Shiga toxin encoding polynucleotide enters anew subspecies or species, the Shiga toxin amino acid sequence ispresumed to be capable of developing slight sequence variations due togenetic drift and/or selective pressure while still maintaining amechanism of cytotoxicity common to members of the Shiga toxin family(see Scheutz F et al., J Clin Microbiol 50: 2951-63 (2012)).

1. De-Immunized, Shiga Toxin a Subunit Effector Polypeptides

In certain embodiments, the Shiga toxin effector polypeptide of thepresent invention is de-immunized, such as, e.g., as compared to awild-type Shiga toxin, wild-type Shiga toxin polypeptide, and/or Shigatoxin effector polypeptide comprising only wild-type polypeptidesequences. The de-immunized, Shiga toxin effector polypeptides of thepresent invention each comprise a disruption of at least one, putative,endogenous, epitope region in order to reduce the antigenic and/orimmunogenic potential of the Shiga toxin effector polypeptide afteradministration of the polypeptide to a chordate. A Shiga toxin effectorpolypeptide and/or Shiga toxin A Subunit polypeptide, whether naturallyoccurring or not, can be de-immunized by a method described herein,described in WO 2015/113005, WO 2015/113007, and/or WO 2016/196344,and/or known to the skilled worker, wherein the resulting moleculeretains one or more Shiga toxin A Subunit functions. Shiga toxineffector polypeptides of the present invention comprise or consistessentially of a polypeptide derived from a Shiga toxin A Subunitdissociated from any form of its native Shiga toxin B Subunit. The Shigatoxin effector polypeptides of the present invention do not comprise thecell-targeting domain of a Shiga toxin B Subunit. Archetypal Shigatoxins naturally target the human cell-surface receptorsglobotriaosylceramide (Gb3, Gb3Cer, or CD77) and globotetraosylceramide(Gb4 or Gb4Cer) via the Shiga toxin B Subunit, which severely limitspotential applications by restricting targeting cell-types andpotentially unwanted targeting of vascular endothelial cells, certainrenal epithelial cells, and/or respiratory epithelial cells (Tesh V etal., Infect Immun 61: 3392-402 (1993); Ling H et al., Biochemistry 37:1777-88 (1998); Bast D et al., Mol Microbiol 32: 953-60 (1999); Rutjes Net al., Kidney Int 62: 832-45 (2002); Shimizu T et al., Microb Pathog43: 88-95 (2007); Pina D et al., Biochim Biophys Acta 1768: 628-36(2007); Shin I et al., BMB Rep 42: 310-4 (2009); Zumbrun S et al.,Infect Immun 4488-99 (2010); Engedal N et al., Microb Biotechnol 4:32-46 (2011); Gallegos K et al., PLoS ONE 7: e30368 (2012); Stahl A etal., PLoS Pathog 11: e1004619 (2015)). Gb3 and Gb4 are a common, neutralsphingolipid present on the extracellular leaflet of cell membranes ofvarious, healthy cell-types, such as polymorphonuclear leukocvtes andhuman endothelial cells from various vascular beds. The cell-targetingmolecules of the present invention do not comprise any polypeptidecomprising or consisting essentially of a functional binding domain of anative Shiga toxin B subunit. Rather, the Shiga toxin effectorpolypeptides of the present invention may be functionally associatedwith heterologous binding regions to effectuate cell targeting.

In certain embodiments, a Shiga toxin effector polypeptide of thepresent invention may comprise or consist essentially of a full-lengthShiga toxin A Subunit (e.g. any one of SEQ ID NOs: 1-18), noting thatnaturally occurring Shiga toxin A Subunits may comprise precursor formscontaining signal sequences of about 22 amino acids at theiramino-terminals which are removed to produce mature Shiga toxin ASubunits and are recognizable to the skilled worker. In otherembodiments, the Shiga toxin effector polypeptide of the inventioncomprises or consists essentially of a truncated Shiga toxin A Subunitwhich is shorter than a full-length Shiga toxin A Subunit, such as,e.g., a truncation known in the art (see e.g., WO 2014/164693; WO2015/113005; WO 2015/113007; WO 2015/138452; WO 2015/191764).

In certain embodiments, the Shiga toxin effector polypeptide of thepresent invention comprises a disruption of an endogenous epitope orepitope region, such as, e.g., a B-cell and/or CD4+ T-cell epitope. Incertain embodiments, the Shiga toxin effector polypeptide of the presentinvention comprises a disruption of at least one, endogenous, epitoperegion described herein, wherein the disruption reduces the antigenicand/or immunogenic potential of the Shiga toxin effector polypeptideafter administration of the polypeptide to a chordate, and wherein theShiga toxin effector polypeptide is capable of exhibiting one or moreShiga toxin A Subunit functions, such as, e.g., a significant level ofShiga toxin cytotoxicity.

The term “disrupted” or “disruption” as used herein with regard to anepitope region refers to the deletion of at least one amino acid residuein an epitope region, inversion of two or more amino acid residues whereat least one of the inverted amino acid residues is in an epitoperegion, insertion of at least one amino acid into an epitope region, anda substitution of at least one amino acid residue in an epitope region.An epitope region disruption by mutation includes amino acidsubstitutions with non-standard amino acids and/or non-natural aminoacids. Epitope regions may alternatively be disrupted by mutationscomprising the modification of an amino acid by the addition of acovalently-linked chemical structure which masks at least one amino acidin an epitope region, see, e.g. PEGylation (see Zhang C et al., BioDrugs26: 209-15 (2012), small molecule adjuvants (Flower D, Expert Opin DrugDiscov 7: 807-17 (2012), and site-specific albumination (Lim S et al., JControl Release 207-93 (2015)).

Certain epitope regions and disruptions are indicated herein byreference to specific amino acid positions of native Shiga toxin ASubunits provided in the Sequence Listing, noting that naturallyoccurring Shiga toxin A Subunits may comprise precursor forms containingsignal sequences of about 22 amino acids at their amino-terminals whichare removed to produce mature Shiga toxin A Subunits and arerecognizable to the skilled worker. Further, certain epitope regiondisruptions are indicated herein by reference to specific amino acids(e.g. S for a serine residue) natively present at specific positionswithin native Shiga toxin A Subunits (e.g. S33 for the serine residue atposition 33 from the amino-terminus) followed by the amino acid withwhich that residue has been substituted in the particular mutation underdiscussion (e.g. S331 represents the amino acid substitution ofisoleucine for serine at amino acid residue 33 from the amino-terminus).

In certain embodiments, the de-immunized, Shiga toxin effectorpolypeptide of the present invention comprises a disruption of at leastone epitope region described in WO 2015/113005, WO 2015/113007 and/or WO2016/196344.

In certain embodiments, the de-immunized, Shiga toxin effectorpolypeptide of the present invention comprises or consists essentiallyof a full-length Shiga toxin A Subunit (e.g. SLT-1A (SEQ ID NO: 1), StxA(SEQ ID NO:2), or SLT-2A (SEQ ID NO:3), or variants thereof (e.g. SEQ IDNOs: 4-18)) comprising at least one disruption of the amino acidsequence selected from the group of natively positioned amino acidsconsisting of: 1-15 of any one of SEQ ID NOs: 1-2 and 4-6; 3-14 of anyone of SEQ ID NOs: 3 and 7-18; 26-37 of any one of SEQ ID NOs: 3 and7-18: 27-37 of any one of SEQ ID NOs: 1-2 and 4-6; 39-48 of any one ofSEQ ID NOs: 1-2 and 4-6: 42-48 of any one of SEQ ID NOs: 3 and 7-18; and53-66 of any one of SEQ ID NOs: 1-18: 94-115 of any one of SEQ ID NOs:1-18; 141-153 of any one of SEQ ID NOs: 1-2 and 4-6: 140-156 of any oneof SEQ ID NOs: 3 and 7-18; 179-190 of any one of SEQ ID NOs: 1-2 and4-6; 179-191 of any one of SEQ ID NOs: 3 and 7-18; 204 of SEQ ID NO:3;205 of any one of SEQ ID NOs: 1-2 and 4-6; and 210-218 of any one of SEQID NOs: 3 and 7-18; 240-260 of any one of SEQ ID NOs: 3 and 7-18;243-257 of any one of SEQ ID NOs: 1-2 and 4-6; 254-268 of any one of SEQID NOs: 1-2 and 4-6; 262-278 of any one of SEQ ID NOs: 3 and 7-18;281-297 of any one of SEQ ID NOs: 3 and 7-18; and 285-293 of any one ofSEQ ID NOs: 1-2 and 4-6; or the equivalent region in a Shiga toxin ASubunit polypeptide, conserved Shiga toxin effector polypeptidesub-region, and/or non-native, Shiga toxin effector polypeptidesequence.

In certain embodiments, the Shiga toxin effector polypeptide of thepresent invention comprises or consists essentially of a truncated Shigatoxin A Subunit. Truncations of Shiga toxin A Subunits might result inthe deletion of an entire epitope region(s) without affecting Shigatoxin effector function(s). The smallest, Shiga toxin A Subunit fragmentshown to exhibit significant enzymatic activity was a polypeptidecomposed of residues 75-247 of StxA (A1-Jaufy A et al., Infect Immun 62:956-60 (1994)). Truncating the carboxy-terminus of SLT-1A, StxA, orSLT-2A to amino acids 1-251 removes two predicted B-cell epitoperegions, two predicted CD4 positive (CD4+) T-cell epitopes, and apredicted, discontinuous, B-cell epitope. Truncating the amino-terminusof SLT-1A, StxA, or SLT-2A to 75-293 removes at least three, predicted,B-cell epitope regions and three predicted CD4+ T-cell epitopes.Truncating both amino- and carboxy-terminals of SLT-1A, StxA, or SLT-2Ato 75-251 deletes at least five, predicted, B-cell epitope regions;four, putative, CD4+ T-cell epitopes; and one, predicted, discontinuous,B-cell epitope.

In certain embodiments, a Shiga toxin effector polypeptide of theinvention may comprise or consist essentially of a full-length ortruncated Shiga toxin A Subunit with at least one mutation, e.g.deletion, insertion, inversion, or substitution, in a provided epitoperegion. In certain further embodiments, the polypeptides comprise adisruption which comprises a deletion of at least one amino acid withinthe epitope region. In certain further embodiments, the polypeptidescomprise a disruption which comprises an insertion of at least one aminoacid within the epitope region. In certain further embodiments, thepolypeptides comprise a disruption which comprises an inversion of aminoacids, wherein at least one inverted amino acid is within the epitoperegion. In certain further embodiments, the polypeptides comprise adisruption which comprises a mutation, such as an amino acidsubstitution to a non-standard amino acid or an amino acid with achemically modified side chain. Numerous examples of single amino acidsubstitutions are provided in the Examples below.

In certain embodiments, the Shiga toxin effector polypeptides of theinvention may comprise or consist essentially of a full-length ortruncated Shiga toxin A Subunit with one or more mutations as comparedto the native sequence which comprises at least one amino acidsubstitution selected from the group consisting of: A, G, V, L, I, P, C,M, F, S, D, N, Q, H, and K. In certain further embodiments, thepolypeptide may comprise or consist essentially of a full-length ortruncated Shiga toxin A Subunit with a single mutation as compared tothe native sequence wherein the substitution is selected from the groupconsisting of: D to A, D to G, D to V, D to L, D to I, D to F, D to S, Dto Q, E to A, E to G, E to V, E to L, E to I, E to F, E to S, E to Q, Eto N, E to D, E to M, E to R, G to A, H to A H to G, H to V, H to L, Hto I, H to F, H to M, K to A, K to G, K to V, K to L, K to I, K to M, Kto H, L to A, L to G, N to A, N to G, N to V, N to L, N to I, N to F, Pto A, P to G, P to F, R to A, R to G, R to V, R to L, R to I, R to F, Rto M, R to Q, R to S, R to K, R to H, S to A, S to G, S to V, S to L, Sto I, S to F, S to M, T to A, T to G, T to V, T to L, T to I, T to F, Tto M, T to S, Y to A, Y to G, Y to V, Y to L, Y to I, Y to F, and Y toM.

In certain embodiments, the Shiga toxin effector polypeptides of theinvention comprise or consist essentially of a full-length or truncatedShiga toxin A Subunit with one or more mutations as compared to thenative amino acid residue sequence which comprises at least one aminoacid substitution of an immunogenic residue and/or within an epitoperegion, wherein at least one substitution occurs at the nativelypositioned group of amino acids selected from the group consisting of: 1of SEQ ID NO: 1 or SEQ ID NO:2; 4 of SEQ ID NO: 1, SEQ ID NO:2, or SEQID NO:3; 8 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 9 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 11 of SEQ ID NO: 1, SEQ ID NO:2, or SEQID NO:3; 33 of SEQ ID NO: 1 or SEQ ID NO:2; 43 of SEQ ID NO:1 or SEQ IDNO:2; 44 of SEQ ID NO:1 or SEQ ID NO:2; 45 of SEQ ID NO:1 or SEQ IDNO:2; 46 of SEQ ID NO: 1 or SEQ ID NO:2; 47 of SEQ ID NO: 1 or SEQ IDNO:2; 48 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 49 of SEQ ID NO:1 or SEQ ID NO:2; 50 of SEQ ID NO: 1 or SEQ ID NO:2; 51 of SEQ ID NO: 1or SEQ ID NO:2; 53 of SEQ ID NO: 1 or SEQ ID NO:2; 54 of SEQ ID NO: 1 orSEQ ID NO:2; 55 of SEQ ID NO: 1 or SEQ ID NO:2; 56 of SEQ ID NO: 1 orSEQ ID NO:2; 57 of SEQ ID NO: 1 or SEQ ID NO:2; 58 of SEQ ID NO: 1, SEQID NO:2, or SEQ ID NO:3; 59 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;60 of SEQ ID NO:1 or SEQ ID NO:2; 61 of SEQ ID NO: 1 or SEQ ID NO:2; 62of SEQ ID NO:1 or SEQ ID NO:2; 84 of SEQ ID NO:1 or SEQ ID NO:2; 88 ofSEQ ID NO:1 or SEQ ID NO:2; 94 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ IDNO:3; 96 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 104 of SEQ ID NO:1 or SEQ ID NO:2; 105 of SEQ ID NO:1 or SEQ ID NO:2; 107 of SEQ ID NO: 1or SEQ ID NO:2; 108 of SEQ ID NO: 1 or SEQ ID NO:2; 109 of SEQ ID NO: 1,SEQ ID NO:2, or SEQ ID NO:3; 110 of SEQ ID NO: 1 or SEQ ID NO:2; 111 ofSEQ ID NO:1 or SEQ ID NO:2; 112 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ IDNO:3; 141 of SEQ ID NO: 1 or SEQ ID NO:2; 147 of SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:3; 154 of SEQ ID NO:1 or SEQ ID NO:2; 179 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3; 180 of SEQ ID NO: 1 or SEQ ID NO:2;181 of SEQ ID NO: 1 or SEQ ID NO:2; 183 of SEQ ID NO: 1, SEQ ID NO:2, orSEQ ID NO:3; 184 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 185 ofSEQ ID NO:1 or SEQ ID NO:2; 186 of SEQ ID NO:1, SEQ ID NO:2, or SEQ IDNO:3; 187 of SEQ ID NO:1 or SEQ ID NO:2; 188 of SEQ ID NO:1 or SEQ IDNO:2; 189 of SEQ ID NO:1 or SEQ ID NO:2; 198 of SEQ ID NO: 1 or SEQ IDNO:2; 204 of SEQ ID NO:3; 205 of SEQ ID NO: 1 or SEQ ID NO:2; 241 of SEQID NO:3; 242 of SEQ ID NO:1 or SEQ ID NO:2; 247 of SEQ ID NO: 1 or SEQID NO:2; 247 of SEQ ID NO:3; 248 of SEQ ID NO:1 or SEQ ID NO:2; 250 ofSEQ ID NO:3; 251 of SEQ ID NO:1 or SEQ ID NO:2; 264 of SEQ ID NO: 1, SEQID NO:2, or SEQ ID NO:3; 265 of SEQ ID NO:1 or SEQ ID NO:2; and 286 ofSEQ ID NO:1 or SEQ ID NO:2.

In certain further embodiments, the Shiga toxin effector polypeptides ofthe invention comprise or consist essentially of a full-length ortruncated Shiga toxin A Subunit with at least one substitution of animmunogenic residue and/or within an epitope region, wherein at leastone amino acid substitution is to a non-conservative amino acid (see,e.g., Table B, infra) relative to a natively occurring amino acidpositioned at one of the following native positions; 1 of SEQ ID NO: 1or SEQ ID NO:2; 4 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 8 of SEQID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 9 of SEQ ID NO: 1, SEQ ID NO:2,or SEQ ID NO:3; 11 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 33 ofSEQ ID NO:1 or SEQ ID NO:2; 43 of SEQ ID NO:1 or SEQ ID NO:2; 44 of SEQID NO: 1 or SEQ ID NO:2; 45 of SEQ ID NO:1 or SEQ ID NO:2; 46 of SEQ IDNO:1 or SEQ ID NO:2; 47 of SEQ ID NO: 1 or SEQ ID NO:2; 48 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 49 of SEQ ID NO: 1 or SEQ ID NO:2; 50 ofSEQ ID NO: 1 or SEQ ID NO:2; 51 of SEQ ID NO: 1 or SEQ ID NO:2; 53 ofSEQ ID NO: 1 or SEQ ID NO:2; 54 of SEQ ID NO: 1 or SEQ ID NO:2; 55 ofSEQ ID NO:1 or SEQ ID NO:2; 56 of SEQ ID NO: 1 or SEQ ID NO:2; 57 of SEQID NO: 1 or SEQ ID NO:2; 58 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ IDNO:3; 59 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 60 of SEQ ID NO:1 or SEQ ID NO:2; 61 of SEQ ID NO: 1 or SEQ ID NO:2; 62 of SEQ ID NO: 1or SEQ ID NO:2; 84 of SEQ ID NO: 1 or SEQ ID NO:2; 88 of SEQ ID NO: 1 orSEQ ID NO:2; 94 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 96 of SEQID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 104 of SEQ ID NO: 1 or SEQ IDNO:2; 105 of SEQ ID NO: 1 or SEQ ID NO:2; 107 of SEQ ID NO: 1 or SEQ IDNO:2; 108 of SEQ ID NO: 1 or SEQ ID NO:2; 109 of SEQ ID NO: 1, SEQ IDNO:2, or SEQ ID NO:3; 110 of SEQ ID NO: 1 or SEQ ID NO:2; 111 of SEQ IDNO:1 or SEQ ID NO:2; 112 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;141 of SEQ ID NO: 1 or SEQ ID NO:2; 147 of SEQ ID NO: 1, SEQ ID NO:2, orSEQ ID NO:3; 154 of SEQ ID NO:1 or SEQ ID NO:2; 179 of SEQ ID NO:1, SEQID NO:2, or SEQ ID NO:3; 180 of SEQ ID NO:1 or SEQ ID NO:2; 181 of SEQID NO: 1 or SEQ ID NO:2; 183 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ IDNO:3; 184 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 185 of SEQ IDNO:1 or SEQ ID NO:2; 186 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3;187 of SEQ ID NO: 1 or SEQ ID NO:2; 188 of SEQ ID NO: 1 or SEQ ID NO:2;189 of SEQ ID NO: 1 or SEQ ID NO:2; 198 of SEQ ID NO: 1 or SEQ ID NO:2;204 of SEQ ID NO:3; 205 of SEQ ID NO: 1 or SEQ ID NO:2; 241 of SEQ IDNO:3; 242 of SEQ ID NO:1 or SEQ ID NO:2; 247 of SEQ ID NO: 1 or SEQ IDNO:2; 247 of SEQ ID NO:3; 248 of SEQ ID NO: 1 or SEQ ID NO:2; 250 of SEQID NO:3; 251 of SEQ ID NO: 1 or SEQ ID NO:2; 264 of SEQ ID NO: 1, SEQ IDNO:2, or SEQ ID NO:3; 265 of SEQ ID NO: 1 or SEQ ID NO:2: and 286 of SEQID NO: 1 or SEQ ID NO:2.

In certain further embodiments, the Shiga toxin effector polypeptides ofthe invention comprise or consist essentially of a full-length ortruncated Shiga toxin A Subunit with at least one amino acidsubstitution selected from the group consisting of K1 to A, G, V, L, I,F, M and H; T4 to A, G, V, L, I, F, M, and S; D6 to A, G, V, L, I, F, S,and Q; S8 to A, G, V, I, L, F, and M; T8 to A, G, V, 1, L, F, M, and S;T9 to A, G, V, I, L, F, M, and S; S9 to A, G, V, L, I, F, and M; K11 toA, G, V, L, I, F, M and H; T12 to A, G, V, 1, L, F, M, and S; S33 to A,G, V, L, 1, F, and M; S43 to A, G, V, L, I, F, and M; G44 to A and L;S45 to A, G, V, L, I, F, and M; T45 to A, G, V, L, 1, F, and M; G46 to Aand P; D47 to A, G, V, L, 1, F, S, and Q; N48 to A, G, V, L, and M; L49to A or G; F50; A51 to V; D53 to A, G, V, L, I, F, S, and Q; V54 to A,G, and L; R55 to A, G, V, L, I, F, M, Q, S, K, and H; G56 to A and P;157 to A, G, M, and F; L57 to A, G, M, and F; D58 to A, G, V, L, I, F,S, and Q; P59 to A, G, and F; E60 to A, G, V, L, I, F, S, Q, N, D, M,and R E61 to A, G, V, L, I, F, S, Q, N, D, M, and R; G62 to A; D94 to A,G, V, L, I, F, S, and Q; R84 to A, G, V, L, I, F, M, Q, S, K, and H; V88to A and G; 188 to A, G, and V; D94; S96 to A, G, V, I, L, F, and M;T104 to A, G, V, I, L, F, M, and S; A105 to L; T107 to A, G, V, I, L, F,M, and S; S107 to A, G, V, L, I, F, and M; L108 to A, G, and M; S109 toA, G, V, I, L, F, and M; T109 to A, G, V, I, L, F, M, and S; G110 to A;D11 to A, G, V, L, I, F, S, and Q; S112 to A, G, V, L, I, F, and M; D141to A, G, V, L, I, F, S, and Q; G147 to A; V154 to A and G; R179 to A, G,V, L, I, F, M, Q, S, K, and H; T180 to A, G, V, L, I, F, M, and S; T181to A, G, V, L, F, M, and S; D183 to A, G, V, L, I, F, S, and Q; D184 toA, G, V, L, I, F, S, and Q; L185 to A, G, and V; S186 to A, G, V, I, L,F, and M; G187 to A; R188 to A, G, V, L, I, F, M, Q, S, K, and H; S189to A, G, V, I, L, F, and M; D197 to A, G, V, L, I, F, S, and Q; D198 toA, G, V, L, I, F, S, and Q; R204 to A, G, V, L, 1, F, M, Q, S, K, and H;R205 to A, G, V, L, I, F, M, Q, S, K and H; C242 to A, G, V, and S; S247to A, G, V, I, L, F, and M; Y247 to A, G, V, L, I, F, and M; R248 to A,G, V, L, I, F, M, Q, S, K, and H; R250 to A, G, V, L, I, F, M, Q, S, K,and H; R251 to A, G, V, L, I, F, M, Q, S, K, and H; C262 to A, G, V, andS; D264 to A, G, V, L, I, F, S, and Q; G264 to A; and T286 to A, G, V,L, I, F, M, and S.

In certain further embodiments, the Shiga toxin effector polypeptides ofthe invention comprise or consist essentially of a full-length ortruncated Shiga toxin A Subunit with at least one of the following aminoacid substitutions K1A, KIM, T41, D6R, S8I, T8V, T91, S91, K11A, K11H,T12K, S331, S33C, S43N, G44L, S45V, S451, T45V, T451, G46P, D47M, D47G,N48V, N48F, L49A, F50T, A51V, D53A, D53N, D53G, V54L, V541, R55A, R55V,R55L, G56P, 157F, 157M, D58A, D58V, D58F, P59A, P59F, E601, E60T, E60R,E61A, E61V, E61L, G62A, R84A, V88A, D94A, S961, T104N, A105L, T107P,L108M, S109V, T109V, G110A, D111T, S112V, D141A, G147A, V154A, R179A,T180G, T1811, D183A, D183G, D184A, D184A, D184F, L185V, L185D, S186A,S186F, G187A, G187T, R188A, R188L, S189A, D198A, R204A, R205A, C242S,S2471, Y247A, R248A R250A, R251A, or D264A, G264A, T286A, and/or T286I.These epitope-disrupting substitutions may be combined to form ade-immunized, Shiga toxin effector polypeptide with multiplesubstitutions per epitope region and/or multiple epitope regionsdisrupted while still retaining Shiga toxin effector function. Forexample, substitutions at the natively positioned K1A, KIM, T41, D6R,S81, T8V, T91, S91, K11A, K11H, T12K, S331, S33C, S43N, G44L, S45V,S451, T45V, T451, G46P, D47M, D47G, N48V, N48F, L49A, F50T, A51V, D53A,D53N, D53G, V54L, V541, R55A, R55V, R55L, G56P, 157F, 157M, D58A, D58V,D58F, P59A, P59F, E601, E60T, E60R, E61A, E61V, E61L, G62A, R84A, V88A,D94A, S961, T104N, A105L, T107P, L108M, S109V, T109V, G110A, DI 1T,S112V, D141A, G147A, V154A, R179A, T180G, T181I, D183A, D183G, D184A,D184A, D184F, L185V, L185D, S186A S186F, G187A, G187T, R188A, R188L,S189A, D198A, R204A, R205A, C242S, S2471, Y247A, R248A, R250A, R251A, orD264A, G264A, T286A, and/or T2861 may be combined, where possible, withsubstitutions at the natively positioned residues K1A, K1M, T4I, D6R,S8I, T8V, T91, S91, K11A, K11H, T12K, S331, S33C, S43N, G44L, S45V,S45I, T45V, T451, G46P, D47M, D47G, N48V, N48F, L49A, F50T, A51V, D53A,D53N, D53G, V54L, V541, R55A, R55V, R55L, G56P, 157F, 157M, D58A, D58V,D58F, P59A, P59F, E601, E60T, E60R, E61A, E61V, E61L, G62A, R84A, V88A,D94A, S961, T104N, A105L, T107P, L108M, S109V, T109V, G110A, D111T,S112V, D41A, G147A, V154A, R179A, T180G, T1811, D183A, D183G, D184A,D184A, D184F, L185V, L185D, S186A, S186F, G187A, G187T, R188A, R188L,S189A, D198A, R204A, R205A, C242S, S247I, Y247A, R248A, R250A, R251A, orD264A, G264A, T286A, and/or T286I to create de-immunized. Shiga toxineffector polypeptides of the invention.

Any of the de-immunized, Shiga toxin effector polypeptide sub-regionsand/or epitope disrupting mutations described herein may be used aloneor in combination with each individual embodiment of the presentinvention, including methods of the present invention.

2. Protease-Cleavage Resistant, Shiga Toxin a Subunit EffectorPolypeptides

In certain embodiments, the Shiga toxin effector polypeptide of thepresent invention comprises (1) a Shiga toxin A1 fragment derived regionhaving a carboxy-terminus and (2) a disrupted furin-cleavage motif atthe carboxy-terminus of the Shiga toxin A1 fragment region. Improvingthe stability of connections between the Shiga toxin component and othercomponents of cell-targeting molecules, e.g., cell-targeting bindingregions, can improve their toxicity profiles after administration toorganisms by reducing non-specific toxicities caused by the breakdown ofthe connection and loss of cell-targeting, such as, e.g., as a result ofproteolysis.

Shiga toxin A Subunits of members of the Shiga toxin family comprise aconserved, furin-cleavage site at the carboxy-terminal of their A1fragment regions important for Shiga toxin function. Furin-cleavage sitemotifs and furin-cleavage sites can be identified by the skilled workerusing standard techniques and/or by using the information herein.

The model of Shiga toxin cytotoxicity is that intracellular proteolyticprocessing of Shiga toxin A Subunits by furin in intoxicated cells isessential for 1) liberation of the A1 fragment from the rest of theShiga holotoxin, 2) escape of the A1 fragment from the endoplasmicreticulum by exposing a hydrophobic domain in the carboxy-terminus ofthe A1 fragment, and 3) enzymatic activation of the A1 fragment (seeJohannes L, Römer W, Nat Rev Microbiol 8: 105-16 (2010)). The efficientliberation of the Shiga toxin A1 fragment from the A2 fragment and therest of the components of the Shiga holotoxin in the endoplasmicreticulum of intoxicated cells is essential for efficient intracellularrouting to the cytosol, maximal enzymatic activity, efficient ribosomeinactivation, and achieving optimal cytotoxicity, i.e. comparable to awild-type Shiga toxin (see e.g. WO 2015/191764 and references therein).

During Shiga toxin intoxication, the A Subunit is proteolyticallycleaved by furin at the carboxy bond of a conserved arginine residue(e.g. the arginine residue at position 251 in StxA and SLT-A variantsand the arginine residue at position 250 in Stx2A and SLT-2A variants).Furin cleavage of Shiga toxin A Subunits occurs in endosomal and/orGolgi compartments. Furin is a specialized serine endoprotease which isexpressed by a wide variety of cell types, in all human tissuesexamined, and by most animal cells. Furin cleaves polypeptidescomprising accessible motifs often centered on the minimal, dibasic,consensus motif R-x-(R/K/x)-R. The A Subunits of members of the Shigatoxin family comprise a conserved, surface-exposed, extended loopstructure (e.g. 242-261 in StxA and SLT-1A, and 241-260 in SLT-2) with aconserved S-R/Y-x-x-R motif which is cleaved by furin. The surfaceexposed, extended loop structure positioned at amino acid residues242-261 in StxA is required for furin-induced cleavage of StxA,including features flanking the minimal, furin-cleavage motif R-x-x-R.

Furin-cleavage motifs and furin-cleavage sites in Shiga toxin A Subunitsand Shiga toxin effector polypeptides can be identified by the skilledworker using standard methods and/or by using the information herein.Furin cleaves the minimal, consensus motif R-x-x-R (Schalken J et al., JClin Invest 80: 1545-9 (1987); Bresnahan P et al., J Cell Biol 111:2851-9 (1990); Hatsuzawa K et al., J Biol Chem 265: 22075-8 (1990); WiseR et al., Proc Natl Acad Sci USA 87: 9378-82 (1990); Molloy S et al., JBiol Chem 267: 16396-402 (1992)). Consistent with this, many furininhibitors comprise peptides comprising the motif R-x-x-R. An example ofa synthetic inhibitor of furin is a molecule comprising the peptideR—V-K-R (Henrich S et al., Nat Struct Biol 10: 520-6 (2003)). Ingeneral, a peptide or protein comprising a surface accessible, dibasicamino acid motif with two positively charged, amino acids separated bytwo amino acid residues may be predicted to be sensitive tofurin-cleavage with cleavage occurring at the carboxy bond of the lastbasic amino acid in the motif.

Consensus motifs in substrates cleaved by furin have been identifiedwith some degree of specificity. A furin-cleavage site motif has beendescribed that comprises a region of twenty, continuous, amino acidresidues, which can be labeled P14 through P6′ (Tian S et al., Int J MolSci 12: 1060-5 (2011)) using the nomenclature described in Schechter I,Berger, A, Biochem Biophys Res Commun 32: 898-902 (1968). According tothis nomenclature, the furin-cleavage site is at the carboxy bond of theamino acid residue designated P1, and the amino acid residues of thefurin-cleavage motif are numbered P2. P3, P4, etc., in the directiongoing toward the amino-terminus from this reference P1 residue. Theamino acid residues of the motif going toward the carboxy-terminus fromthe P1 reference residue are numbered with the prime notation P2′, P3′,P4′, etc. Using this nomenclature, the P6 to P2′ region delineates thecore substrate of the furin cleavage motif which is bound by theenzymatic domain of furin. The two flanking regions P14 to P7 and P3′ toP6′ are often rich in polar, amino acid residues to increase theaccessibility to the core furin cleavage site located between them.

A general, furin-cleavage site is often described by the consensus motifR-x-x-R which corresponds to P4-P3-P2-P1; where “R” represents anarginine residue (see Table A, supra), a dash “-” represents a peptidebond, and a lowercase “x” represents any amino acid residue. However,other residues and positions may help to further define furin-cleavagemotifs. A slightly more refined furin-cleavage site, consensus motif isoften reported as the consensus motif R-x-[K/R]-R (where a forward slash“/” means “or” and divides alternative amino acid residues at the sameposition), which corresponds to P4-P3-P2-P1, because it was observedthat furin has a strong preference for cleaving substrates containingthis motif.

In addition to the minimal, furin-cleavage site R-x-x-R, a larger,furin-cleavage motif has been described with certain amino acid residuepreferences at certain positions. By comparing various known furinsubstrates, certain physicochemical properties have been characterizedfor the amino acid residues in a amino acid residue long, furin-cleavagesite motif. The P6 to P2′ region of the furin-cleavage motif delineatesthe core furin-cleavage site which physically interacts with theenzymatic domain of furin. The two flanking regions P14 to P7 and P3′ toP6′ are often hydrophilic being rich in polar, amino acid residues toincrease the surface accessibility of the core furin-cleavage sitelocated between them.

In general, the furin-cleavage motif region from position P5 to P1 tendsto comprise amino acid residues with a positive charge and/or highisoelectric points. In particular, the P1 position, which marks theposition of furin proteolysis, is generally occupied by an arginine butother positively charged, amino acid residues may occur in thisposition. Positions P2 and P3 tend to be occupied by flexible, aminoacid residues, and in particular P2 tends to be occupied by arginine,lysine, or sometimes by very small and flexible amino acid residues likeglycine. The P4 position tends to be occupied by positively charged,amino acid residues in furin substrates. However, if the P4 position isoccupied by an aliphatic, amino acid residue, then the lack of apositively charged, functional group can be compensated for by apositively charged residue located at position(s) P5 and/or P6.Positions P1′ and P2′ are commonly occupied by aliphatic and/orhydrophobic amino acid residues, with the P1 position most commonlybeing occupied by a serine.

The two, hydrophilic, flanking regions tend to be occupied by amino acidresidues which are polar, hydrophilic, and have smaller amino acidfunctional groups, however, in certain verified furin substrates, theflanking regions do not contain any hydrophilic, amino acid residues(see Tian S, Biochem Insights 2: 9-20 (2009)).

The twenty amino acid residue, furin-cleavage motif and furin-cleavagesite found in native, Shiga toxin A Subunits at the junction between theShiga toxin A1 fragment and A2 fragment is well characterized in certainShiga toxins. For example in StxA (SEQ ID NO:2) and SLT-1A (SEQ ID NO:1), this furin-cleavage motif is natively positioned from L238 to F257,and in SLT-2A (SEQ ID NO:3), this furin-cleavage motif is nativelypositioned from V237 to Q256. Based on amino acid homology, experiment,and/or furin-cleavage assays described herein, the skilled worker canidentify furin-cleavage motifs in other native, Shiga toxin A Subunitsor Shiga toxin effector polypeptides, where the motifs are actualfurin-cleavage motifs or are predicted to result in the production of A1and A2 fragments after furin cleavage of those molecules within aeukaryotic cell.

In certain embodiments, the Shiga toxin effector polypeptide of thepresent invention comprises (1) a Shiga toxin A1 fragment derivedpolypeptide having a carboxy-terminus and (2) a disrupted furin-cleavagemotif at the carboxy-terminus of the Shiga toxin A1 fragment derivedpolypeptide. The carboxy-terminus of a Shiga toxin A1 fragment derivedpolypeptide may be identified by the skilled worker by using techniquesknown in the art, such as, e.g., by using protein sequence alignmentsoftware to identify (i) a furin-cleavage motif conserved with anaturally occurring Shiga toxin, (ii) a surface exposed, extended loopconserved with a naturally occurring Shiga toxin, and/or (iii) a stretchof amino acid residues which are predominantly hydrophobic (i.e. ahydrophobic “patch”) that may be recognized by the ERAD system

A protease-cleavage resistant, Shiga toxin effector polypeptide of thepresent invention (1) may be completely lacking any furin-cleavage motifat a carboxy-terminus of its Shiga toxin A1 fragment region and/or (2)comprise a disrupted furin-cleavage motif at the carboxy-terminus of itsShiga toxin A1 fragment region and/or region derived from thecarboxy-terminus of a Shiga toxin A1 fragment. A disruption of afurin-cleavage motif includes various alterations to an amino acidresidue in the furin-cleavage motif, such as, e.g., a post-translationmodification(s), an alteration of one or more atoms in an amino acidfunctional group, the addition of one or more atoms to an amino acidfunctional group, the association to a non-proteinaceous moiety(ies),and/or the linkage to an amino acid residue, peptide, polypeptide suchas resulting in a branched proteinaceous structure.

Protease-cleavage resistant, Shiga toxin effector polypeptides may becreated from a Shiga toxin effector polypeptide and/or Shiga toxin ASubunit polypeptide, whether naturally occurring or not, using a methoddescribed herein, described in WO 2015/191764 and WO 2016/196344, and/orknown to the skilled worker, wherein the resulting molecule stillretains one or more Shiga toxin A Subunit functions.

For purposes of the present invention with regard to a furin-cleavagesite or furin-cleavage motif, the term “disruption” or “disrupted”refers to an alteration from the naturally occurring furin-cleavage siteand/or furin-cleavage motif, such as, e.g., a mutation, that results ina reduction in furin-cleavage proximal to the carboxy-terminus of aShiga toxin A1 fragment region, or identifiable region derived thereof,as compared to the furin-cleavage of a wild-type Shiga toxin A Subunitor a polypeptide derived from a wild-type Shiga toxin A Subunitcomprising only wild-type polypeptide sequences. An alteration to anamino acid residue in the furin-cleavage motif includes a mutation inthe furin-cleavage motif, such as, e.g., a deletion, insertion,inversion, substitution, and/or carboxy-terminal truncation of thefurin-cleavage motif, as well as a post-translation modification, suchas, e.g., as a result of glycosylation, albumination, and the like whichinvolve conjugating or linking a molecule to the functional group of anamino acid residue. Because the furin-cleavage motif is comprised ofabout twenty, amino acid residues, in theory, alterations,modifications, mutations, deletions, insertions, and/or truncationsinvolving one or more amino acid residues of any one of these twentypositions might result in a reduction of furin-cleavage sensitivity(Tian S et al., Sci Rep 2: 261 (2012)). The disruption of afurin-cleavage site and/or furin-cleavage motif may or may not increaseresistance to cleavage by other proteases, such as, e.g., trypsin andextracellular proteases common in the vascular system of mammals. Theeffects of a given disruption to cleavage sensitivity of a givenprotease may be tested by the skilled worker using techniques known inthe art.

For purposes of the present invention, a “disrupted furin-cleavagemotif” is furin-cleavage motif comprising an alteration to one or moreamino acid residues derived from the 20 amino acid residue regionrepresenting a conserved, furin-cleavage motif found in native, Shigatoxin A Subunits at the junction between the Shiga toxin A1 fragment andA2 fragment regions and positioned such that furin cleavage of a Shigatoxin A Subunit results in the production of the A1 and A2 fragments;wherein the disrupted furin-cleavage motif exhibits reduced furincleavage in an experimentally reproducible way as compared to areference molecule comprising a wild-type, Shiga toxin A1 fragmentregion fused to a carboxy-terminal polypeptide of a size large enough tomonitor furin cleavage using the appropriate assay known to the skilledworker and/or described herein.

Examples of types of mutations which can disrupt a furin-cleavage siteand furin-cleavage motif are amino acid residue deletions, insertions,truncations, inversions, and/or substitutions, including substitutionswith non-standard amino acids and/or non-natural amino acids. Inaddition, furin-cleavage sites and furin-cleavage motifs can bedisrupted by mutations comprising the modification of an amino acid bythe addition of a covalently-linked structure which masks at least oneamino acid in the site or motif, such as, e.g., as a result ofPEGylation, the coupling of small molecule adjuvants, and/orsite-specific albumination.

If a furin-cleavage motif has been disrupted by mutation and/or thepresence of non-natural amino acid residues, certain disruptedfurin-cleavage motifs may not be easily recognizable as being related toany furin-cleavage motif; however, the carboxy-terminus of the Shigatoxin A1 fragment derived region will be recognizable and will definewhere the furin-cleavage motif would be located were it not disrupted.For example, a disrupted furin-cleavage motif may comprise less than thetwenty, amino acid residues of the furin-cleavage motif due to acarboxy-terminal truncation as compared to a Shiga toxin A Subunitand/or Shiga toxin A1 fragment.

In certain embodiments, the Shiga toxin effector polypeptide of thepresent invention comprises (1) a Shiga toxin A1 fragment derivedpolypeptide having a carboxy-terminus and (2) a disrupted furin-cleavagemotif at the carboxy-terminus of the Shiga toxin A1 fragment polypeptideregion; wherein the Shiga toxin effector polypeptide (and anycell-targeting molecule comprising it) is more furin-cleavage resistantas compared to a reference molecule, such as, e.g., a wild-type Shigatoxin polypeptide comprising the carboxy-terminus of an A1 fragmentand/or the conserved, furin-cleavage motif between A1 and A2 fragments.For example, a reduction in furin cleavage of one molecule compared to areference molecule may be determined using an in vitro, furin-cleavageassay described in the Examples below, conducted using the sameconditions, and then performing a quantitation of the band density ofany fragments resulting from cleavage to quantitatively measure inchange in furin cleavage.

In certain embodiments, the Shiga toxin effector polypeptide is moreresistant to furin-cleavage in vitro and/or in vivo as compared to awild-type, Shiga toxin A Subunit.

In general, the protease-cleavage sensitivity of a cell-targetingmolecule of the present invention is tested by comparing it to the samemolecule having its furin-cleavage resistant, Shiga toxin effectorpolypeptide replaced with a wild-type, Shiga toxin effector polypeptidecomprising a Shiga toxin A1 fragment. In certain embodiments, themolecules of the present invention comprising a disrupted furin-cleavagemotif exhibits a reduction in in vitro furin cleavage of 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 97%, 98% or greater compared to a referencemolecule comprising a wild-type, Shiga toxin A1 fragment fused at itscarboxy-terminus to a peptide or polypeptide, such as, e.g., thereference molecule SLT-1A-WT::scFv1::C2 (SEQ ID NO:278) described in theExamples below.

Several furin-cleavage motif disruptions have been described. Forexample, mutating the two conserved arginines to alanines in the minimalR-x-x-R motif completely blocked processing by furin and/or furin-likeproteases (see e.g. Duda A et al., J Virology 78: 13865-70 (2004)).Because the furin-cleavage site motif is comprised of about twenty aminoacid residues, in theory, certain mutations involving one or more of anyone of these twenty, amino acid residue positions might abolish furincleavage or reduce furin cleavage efficiency (see e.g. Tian S et al.,Sci Rep 2: 261 (2012)).

In certain embodiments, the molecules of the present invention comprisea Shiga toxin effector polypeptide derived from at least one A Subunitof a member of the Shiga toxin family wherein the Shiga toxin effectorpolypeptide comprises a disruption in one or more amino acids derivedfrom the conserved, highly accessible, protease-cleavage sensitive loopof Shiga toxin A Subunits. For example, in StxA and SLT-1A, this highlyaccessible, protease-sensitive loop is natively positioned from aminoacid residues 242 to 261, and in SLT-2A, this conserved loop is nativelypositioned from amino acid residues 241 to 260. Based on polypeptidesequence homology, the skilled worker can identify this conserved,highly accessible loop structure in other Shiga toxin A Subunits.Certain mutations to the amino acid residues in this loop can reduce theaccessibility of certain amino acid residues within the loop toproteolytic cleavage and this might reduce furin-cleavage sensitivity.

In certain embodiments, a molecule of the present invention comprises aShiga toxin effector polypeptide comprising a disrupted furin-cleavagemotif comprising a mutation in the surface-exposed, protease sensitiveloop conserved among Shiga toxin A Subunits. In certain furtherembodiments, a molecule of the present invention comprises a Shiga toxineffector polypeptide comprising a disrupted furin-cleavage motifcomprising a mutation in this protease-sensitive loop of Shiga toxin ASubunits, the mutation which reduce the surface accessibility of certainamino acid residues within the loop such that furin-cleavage sensitivityis reduced.

In certain embodiments, the disrupted furin-cleavage motif of a Shigatoxin effector polypeptide of the present invention comprises adisruption in terms of existence, position, or functional group of oneor both of the consensus amino acid residues P1 and P4, such as, e.g.,the amino acid residues in positions I and 4 of the minimalfurin-cleavage motif R/Y-x-x-R. For example, mutating one or both of thetwo arginine residues in the minimal, furin consensus site R-x-x-R toalanine will disrupt a furin-cleavage motif and prevent furin-cleavageat that site. Similarly, amino acid residue substitutions of one or bothof the arginine residues in the minimal furin-cleavage motif R-x-x-R toany non-conservative amino acid residue known to the skilled worker willreduced the furin-cleavage sensitivity of the motif. In particular,amino acid residue substitutions of arginine to any non-basic amino acidresidue which lacks a positive charge, such as, e.g., A, G, P, S, T, D,E, Q, N, C, I, L, M, V, F, W, and Y, will result in a disruptedfurin-cleavage motif.

In certain embodiments, the disrupted furin-cleavage motif of a Shigatoxin effector polypeptide of the present invention comprises adisruption in the spacing between the consensus amino acid residues P4and P1 in terms of the number of intervening amino acid residues beingother than two, and, thus, changing either P4 and/or P1 into a differentposition and eliminating the P4 and/or PI designations. For example,deletions within the furin-cleavage motif of the minimal furin-cleavagesite or the core, furin-cleavage motif will reduce the furin-cleavagesensitivity of the furin-cleavage motif.

In certain embodiments, the disrupted furin-cleavage motif comprises oneor more amino acid residue substitutions, as compared to a wild-type,Shiga toxin A Subunit. In certain further embodiments, the disruptedfurin-cleavage motif comprises one or more amino acid residuesubstitutions within the minimal furin-cleavage site R/Y-x-x-R, such as,e.g., for StxA and SLT-1A derived Shiga toxin effector polypeptides, thenatively positioned amino acid residue R248 substituted with anynon-positively charged, amino acid residue and/or R251 substituted withany non-positively charged, amino acid residue; and for SLT-2A derivedShiga toxin effector polypeptides, the natively positioned amino acidresidue Y247 substituted with any non-positively charged, amino acidresidue and/or R250 substituted with any non-positively charged, aminoacid residue.

In certain embodiments, the disrupted furin-cleavage motif comprises anun-disrupted, minimal furin-cleavage site R/Y-x-x-R but insteadcomprises a disrupted flanking region, such as, e.g., amino acid residuesubstitutions in one or more amino acid residues in the furin-cleavagemotif flanking regions natively position at, e.g., 241-247 and/or252-259. In certain further embodiments, the disrupted furin cleavagemotif comprises a substitution of one or more of the amino acid residueslocated in the P1-P6 region of the furin-cleavage motif; mutating P1′ toa bulky amino acid, such as, e.g., R, W, Y, F, and H; and mutating P2′to a polar and hydrophilic amino acid residue; and substituting one ormore of the amino acid residues located in the P1′-P6′ region of thefurin-cleavage motif with one or more bulky and hydrophobic amino acidresidues

In certain embodiments, the disruption of the furin-cleavage motifcomprises a deletion, insertion, inversion, and/or mutation of at leastone amino acid residue within the furin-cleavage motif. In certainembodiments, a protease-cleavage resistant, Shiga toxin effectorpolypeptide of the present invention may comprise a disruption of theamino acid sequence natively positioned at 248-251 of the A Subunit ofShiga toxin (SEQ ID NOs: 1-2 and 4-6), at 247-250 of the A Subunit ofShiga-like toxin 2 (SEQ ID NOs: 3 and 7-18), or at the equivalentposition in a conserved Shiga toxin effector polypeptide and/ornon-native Shiga toxin effector polypeptide sequence. In certain furtherembodiments, protease-cleavage resistant, Shiga toxin effectorpolypeptides comprise a disruption which comprises a deletion of atleast one amino acid within the furin-cleavage motif. In certain furtherembodiments, protease-cleavage resistant, Shiga toxin effectorpolypeptides comprise a disruption which comprises an insertion of atleast one amino acid within the protease-cleavage motif region. Incertain further embodiments, the protease-cleavage resistant, Shigatoxin effector polypeptides comprise a disruption which comprises aninversion of amino acids, wherein at least one inverted amino acid iswithin the protease motif region. In certain further embodiments, theprotease-cleavage resistant, Shiga toxin effector polypeptides comprisea disruption which comprises a mutation, such as an amino acidsubstitution to a non-standard amino acid or an amino acid with achemically modified side chain. Examples of single amino acidsubstitutions are provided in the Examples below.

In certain embodiments of the molecules of the present invention, thedisrupted furin-cleavage motif comprises the deletion of nine, ten,eleven, or more of the carboxy-terminal amino acid residues within thefurin-cleavage motif. In these embodiments, the disrupted furin-cleavagemotif will not comprise a furin-cleavage site or a minimalfurin-cleavage motif. In other words, certain embodiments lack afurin-cleavage site at the carboxy-terminus of the A1 fragment region.

In certain embodiments, the disrupted furin-cleavage motif comprisesboth an amino acid residue deletion and an amino acid residuesubstitution as compared to a wild-type, Shiga toxin A Subunit. Incertain further embodiments, the disrupted furin-cleavage motifcomprises one or more amino acid residue deletions and substitutionswithin the minimal furin-cleavage site R/Y-x-x-R, such as, e.g., forStxA and SLT-1A derived Shiga toxin effector polypeptides, the nativelypositioned amino acid residue R248 substituted with any non-positivelycharged, amino acid residue and/or R251 substituted with anynon-positively charged, amino acid residue; and for SLT-2A derived Shigatoxin effector polypeptides, the natively positioned amino acid residueY247 substituted with any non-positively charged, amino acid residueand/or R250 substituted with any non-positively charged, amino acidresidue.

In certain embodiments, the disrupted furin-cleavage motif comprises anamino acid residue deletion and an amino acid residue substitution aswell as a carboxy-terminal truncation as compared to a wild-type, Shigatoxin A Subunit. In certain further embodiments, the disruptedfurin-cleavage motif comprises one or more amino acid residue deletionsand substitutions within the minimal furin-cleavage site R/Y-x-x-R, suchas, e.g., for StxA and SLT-1A derived Shiga toxin effector polypeptides,the natively positioned amino acid residue R248 substituted with anynon-positively charged, amino acid residue and/or R251 substituted withany non-positively charged, amino acid residue, and for SLT-2A derivedShiga toxin effector polypeptides, the natively positioned amino acidresidue Y247 substituted with any non-positively charged, amino acidresidue and/or R250 substituted with any non-positively charged, aminoacid residue.

In certain further embodiments, the disrupted furin-cleavage motifcomprises both an amino acid substitution within the minimalfurin-cleavage site R/Y-x-x-R and a carboxy-terminal truncation ascompared to a wild-type, Shiga toxin A Subunit, such as, e.g., for StxAand SLT-1A derived Shiga toxin effector polypeptides, truncations endingat the natively amino acid position 249, 250, 251, 252, 253, 254, 255,256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269,270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283,284, 285, 286, 287, 288, 289, 290, 291, or greater and comprising thenatively positioned amino acid residue R248 and/or R251 substituted withany non-positively charged, amino acid residue where appropriate; andfor SLT-2A derived Shiga toxin effector polypeptides, truncations endingat the natively amino acid position 248, 249, 250, 251, 252, 253, 254,255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268,269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282,283, 284, 285, 286, 287, 288, 289, 290, 291, or greater and comprisingthe natively positioned amino acid residue Y247 and/or R250 substitutedwith any non-positively charged, amino acid residue where appropriate.

In certain embodiments, the disrupted furin-cleavage motif comprises aninsertion of one or more amino acid residues as compared to a wild-type,Shiga toxin A Subunit as long as the inserted amino residue(s) does notcreate a de novo furin-cleavage site. In certain embodiments, theinsertion of one or more amino acid residues disrupts the naturalspacing between the arginine residues in the minimal, furin-cleavagesite R/Y-x-x-R, such as, e.g., StxA and SLT-1A derived polypeptidescomprising an insertion of one or more amino acid residues at 249 or 250and thus between R248 and R251; or SLT-2A derived polypeptidescomprising an insertion of one or more amino acid residues at 248 or 249and thus between Y247 and R250.

In certain embodiments, the disrupted furin-cleavage motif comprisesboth an amino acid residue insertion and a carboxy-terminal truncationas compared to a wild-type, Shiga toxin A Subunit. In certainembodiments, the disrupted furin-cleavage motif comprises both an aminoacid residue insertion and an amino acid residue substitution ascompared to a wild-type, Shiga toxin A Subunit. In certain embodiments,the disrupted furin-cleavage motif comprises both an amino acid residueinsertion and an amino acid residue deletion as compared to a wild-type,Shiga toxin A Subunit.

In certain embodiments, the disrupted furin-cleavage motif comprises anamino acid residue deletion, an amino acid residue insertion, and anamino acid residue substitution as compared to a wild-type, Shiga toxinA Subunit.

In certain embodiments, the disrupted furin-cleavage motif comprises anamino acid residue deletion, insertion, substitution, andcarboxy-terminal truncation as compared to a wild-type, Shiga toxin ASubunit.

In certain embodiments, the Shiga toxin effector polypeptide comprisinga disrupted furin-cleavage motif is directly fused by a peptide bond toa molecular moiety comprising an amino acid, peptide, and/or polypeptidewherein the fused structure involves a single, continuous polypeptide.In these fusion embodiments, the amino acid sequence following thedisrupted furin-cleavage motif should not create a de novo,furin-cleavage site at the fusion junction.

Any of the above protease-cleavage resistant, Shiga toxin effectorpolypeptide disrupted furin-cleavage motifs may be used alone or incombination with each individual embodiment of the present invention,including methods of the present invention.

3. T-Cell Hyper-Immunized, Shiga Toxin A Subunit Effector Polypeptides

In certain embodiments, the Shiga toxin effector polypeptide of thepresent invention comprises an embedded or inserted epitope-peptide. Incertain further embodiments, the epitope-peptide is a heterologous,T-cell epitope-peptide, such as, e.g., an epitope consideredheterologous to Shiga toxin A Subunits. In certain further embodiments,the epitope-peptide is a CD8+ T-cell epitope. In certain furtherembodiments, the CD8+ T-cell epitope-peptide has a binding affinity to aMHC class I molecule characterized by a dissociation constant (K_(D)) of10⁻⁴ molar or less and/or the resulting MHC class I-epitope-peptidecomplex has a binding affinity to a T-cell receptor (TCR) characterizedby a dissociation constant (K_(D)) of 10⁻⁴ molar or less.

In certain embodiments, the Shiga toxin effector polypeptide of thepresent invention comprises an embedded or inserted, heterologous,T-cell epitope, such as, e.g., a human CD8+ T-cell epitope. In certainfurther embodiments, the heterologous, T-cell epitope is embedded orinserted so as to disrupt an endogenous epitope or epitope region (e.g.a B-cell epitope and/or CD4+ T-cell epitope) identifiable in a naturallyoccurring Shiga toxin polypeptide or parental Shiga toxin effectorpolypeptide from which the Shiga toxin effector polypeptide of thepresent invention is derived.

For certain embodiments of the present invention, the Shiga toxineffector polypeptide (and any cell-targeting molecule comprising it) isCD8+ T-cell hyper-immunized, such as, e.g., as compared to a wild-typeShiga toxin polypeptide. The CD8+ T-cell hyper-immunized, Shiga toxineffector polypeptides of the present invention each comprise a T-cellepitope-peptide. Hyper-immunized, Shiga toxin effector polypeptides canbe created from Shiga toxin effector polypeptides and/or Shiga toxin ASubunit polypeptides, whether naturally occurring or not, using a methoddescribed herein, described in WO 2015/113007, and/or known to theskilled worker, wherein the resulting molecule still retains one or moreShiga toxin A Subunit functions.

For purposes of the claimed invention, a T-cell epitope is a molecularstructure which is comprised by an antigenic peptide and can berepresented by a linear, amino acid sequence. Commonly, T-cell epitopesare peptides of sizes of eight to eleven amino acid residues (TownsendA, Bodmer H, Annu Rev Immunol 7: 601-24 (1989)); however, certain T-cellepitope-peptides have lengths that are smaller than eight or larger thaneleven amino acids long (see e.g. Livingstone A, Fathman C, Annu RevImmunol 5: 477-501 (1987); Green K et al., Eur J Immunol 34: 2510-9(2004)). In certain embodiments, the embedded or inserted epitope is atleast seven amino acid residues in length. In certain embodiments, theembedded or inserted epitope is bound by a TCR with a binding affinitycharacterized by a K_(D) less than 10 mM (e.g. 1-100 pM) as calculatedusing the formula in Stone J et al., Immunology 126: 165-76 (2009).However, it should be noted that the binding affinity within a givenrange between the MHC-epitope and TCR may not correlate withantigenicity and/or immunogenicity (see e.g. A1-Ramadi B et al., JImmunol 155: 662-73 (1995)), such as due to factors like MHC-peptide-TCRcomplex stability, MHC-peptide density and MHC-independent functions ofTCR cofactors such as CD8 (Valitutti S et al., J Exp Med 183: 1917-21(1996); Baker B et al., Immunity 13: 475-84 (2000); Hornell T et al., JImmunol 170: 4506-14 (2003); Faroudi M et al., Proc Natl Acad Sci USA100: 14145-50 (2003); Woolridge L et al., J Immunol 171: 6650-60 (2003);Purbhoo M et al., Nat Immunol 5: 524-30 (2004)).

A heterologous, T-cell epitope is an epitope not already present in awild-type Shiga toxin A Subunit; a naturally occurring Shiga toxin ASubunit; and/or a parental, Shiga toxin effector polypeptide used as asource polypeptide for modification by a method described herein,described in WO 2015/113007 and/or WO 2016/196344, and/or known to theskilled worker.

A heterologous, T-cell epitope-peptide may be incorporated into a sourcepolypeptide via numerous methods known to the skilled worker, including,e.g., the processes of creating one or more amino acid substitutionswithin the source polypeptide, fusing one or more amino acids to thesource polypeptide, inserting one or more amino acids into the sourcepolypeptide, linking a peptide to the source polypeptide, and/or acombination of the aforementioned processes. The result of such a methodis the creation of a modified variant of the source polypeptide whichcomprises one or more embedded or inserted, heterologous, T-cellepitope-peptides.

T-cell epitopes may be chosen or derived from a number of sourcemolecules for use in the present invention. T-cell epitopes may becreated or derived from various naturally occurring proteins. T-cellepitopes may be created or derived from various naturally occurringproteins foreign to mammals, such as, e.g., proteins of microorganisms.T-cell epitopes may be created or derived from mutated human proteinsand/or human proteins aberrantly expressed by malignant human cells.T-cell epitopes may be synthetically created or derived from syntheticmolecules (see e.g., Carbone F et al., J Exp Med 167: 1767-9 (1988); DelVal M et al., 0.1 Virol 65: 3641-6 (1991); Appella E et al., Biomed PeptProteins Nucleic Acids 1: 177-84 (1995); Perez S et al., Cancer 116:2071-80 (2010)).

Although any T-cell epitope-peptide is contemplated as being used as aheterologous, T-cell epitope of the present invention, certain epitopesmay be selected based on desirable properties. One objective of thepresent invention is to create CD8+ T-cell hyper-immunized, Shiga toxineffector polypeptides for administration to vertebrates, meaning thatthe heterologous, T-cell epitope is highly immunogenic and can elicitrobust immune responses in vivo when displayed complexed with a MHCclass I molecule on the surface of a cell. In certain embodiments, theShiga toxin effector polypeptide of the present invention comprises oneor more, embedded or inserted, heterologous, T-cell epitopes which areCD8+ T-cell epitopes. A Shiga toxin effector polypeptide of the presentinvention that comprises a heterologous, CD8+ T-cell epitope isconsidered a CD8+ T-cell hyper-immunized, Shiga toxin effectorpolypeptide.

T-cell epitope components of the present invention may be chosen orderived from a number of source molecules already known to be capable ofeliciting a vertebrate immune response. T-cell epitopes may be derivedfrom various naturally occurring proteins foreign to vertebrates, suchas, e.g., proteins of pathogenic microorganisms and non-self, cancerantigens. In particular, infectious microorganisms may contain numerousproteins with known antigenic and/or immunogenic properties. Further,infectious microorganisms may contain numerous proteins with knownantigenic and/or immunogenic sub-regions or epitopes.

For example, the proteins of intracellular pathogens with mammalianhosts are sources for T-cell epitopes. There are numerous intracellularpathogens, such as viruses, bacteria, fungi, and single-cell eukaryotes,with well-studied antigenic proteins or peptides. T-cell epitopes can beselected or identified from human viruses or other intracellularpathogens, such as, e.g., bacteria like mycobacterium, fungi liketoxoplasmae, and protists like trypanosomes.

For example, there are many immunogenic, viral peptide components ofviral proteins from viruses that are infectious to humans. Numerous,human T-cell epitopes have been mapped to peptides within proteins frominfluenza A viruses, such as peptides in the proteins HA glycoproteinsFE 17. S139/1, CH65, C05, hemagglutinin 1 (HA1), hemagglutinin 2 (HA2),nonstructural protein 1 and 2 (NS1 and NS 2), matrix protein 1 and 2 (M1and M2), nucleoprotein (NP), neuraminidase (NA)), and many of thesepeptides have been shown to elicit human immune responses, such as byusing ex vivo assay. Similarly, numerous, human T-cell epitopes havebeen mapped to peptide components of proteins from humancytomegaloviruses (HCMV), such as peptides in the proteins pp65 (UL83),UL128-131, immediate-early 1 (IE-1; UL123), glycoprotein B, tegumentproteins, and many of these peptides have been shown to elicit humanimmune responses, such as by using ex vivo assays.

Another example is there are many immunogenic, cancer antigens inhumans. The CD8+ T-cell epitopes of cancer and/or tumor cell antigenscan be identified by the skilled worker using techniques known in theart, such as, e.g., differential genomics, differential proteomics,immunoproteomics, prediction then validation, and genetic approacheslike reverse-genetic transfection (see e.g., Admon A et al., Mol CellProteomics 2: 388-98 (2003); Purcell A, Gorman J, Mol Cell Proteomics 3:193-208 (2004); Comber J, Philip R, Ther Adv Vaccines 2: 77-89 (2014)).There are many antigenic and/or immunogenic T-cell epitopes alreadyidentified or predicted to occur in human cancer and/or tumor cells. Forexample, T-cell epitopes have been predicted in human proteins commonlymutated or overexpressed in neoplastic cells, such as, e.g., ALK. CEA,N-acetylglucosaminyl-transferase V (GnT-V), HCA587, HER-2/neu, MAGE,Melan-A/MART-1, MUC-1, p53, and TRAG-3 (see e.g., van der Bruggen P etal., Science 254: 1643-7 (1991); Kawakami Y et al., J Exp Med 180:347-52 (1994); Fisk B et al., J Exp Med 181: 2109-17 (1995); Guilloux Yet al., J Exp Med 183: 1173 (1996); Skipper J et al., J Exp Med 183: 527(1996); Brossart P et al., 93: 4309-17 (1999); Kawashima I et al.,Cancer Res 59: 431-5 (1999); Papadopoulos K et al., Clin Cancer Res 5:2089-93 (1999); Zhu B et al., Clin Cancer Res 9: 1850-7 (2003); Li B etal., Clin Exp Immunol 140: 310-9 (2005); Ait-Tahar K et al., Int JCancer 118: 688-95 (2006); Akiyama Y et al., Cancer Immunol Immunother61: 2311-9 (2012)). In addition, synthetic variants of T-cell epitopesfrom human cancer cells have been created (see e.g., Lazoura E,Apostolopoulos V, Curr Med Chem 12: 629-39 (2005); Douat-Casassus C etal., J Med Chem 50: 1598-609 (2007)).

While any T-cell epitope may be used in the polypeptides and moleculesof the present invention, certain T-cell epitopes may be preferred basedon their known and/or empirically determined characteristics. Forexample, in many species, the MHC alleles in its genome encode multipleMHC-I molecular variants. Because MHC class I protein polymorphisms canaffect antigen-MHC class I complex recognition by CD8+ T-cells, T-cellepitopes may be chosen for use in the present invention based onknowledge about certain MHC class I polymorphisms and/or the ability ofcertain antigen-MHC class I complexes to be recognized by T-cells havingdifferent genotypes.

There are well-defined peptide-epitopes that are known to beimmunogenic, MHC class I restricted, and/or matched with a specifichuman leukocyte antigen (HLA) variant(s). For applications in humans orinvolving human target cells, HLA-class I-restricted epitopes can beselected or identified by the skilled worker using standard techniquesknown in the art. The ability of peptides to bind to human MHC class Imolecules can be used to predict the immunogenic potential of putativeT-cell epitopes. The ability of peptides to bind to human MHC class Imolecules can be scored using software tools. T-cell epitopes may bechosen for use as a heterologous, T-cell epitope component of thepresent invention based on the peptide selectivity of the HLA variantsencoded by the alleles more prevalent in certain human populations. Forexample, the human population is polymorphic for the alpha chain of MHCclass I molecules due to the varied alleles of the HLA genes fromindividual to individual. In certain T-cell epitopes may be moreefficiently presented by a specific HLA molecule, such as, e.g., thecommonly occurring HLA variants encoded by the HLA-A allele groupsHLA-A2 and HLA-A3.

When choosing T-cell epitopes for use as a heterologous, T-cell epitopecomponent of the present invention, multiple factors may be consideredthat can influence epitope generation and transport to receptive MHCclass I molecules, such as, e.g., the presence and epitope specificityof the following factors in the target cell: proteasome, ERAAP/ERAP1,tapasin, and TAPs.

When choosing T-cell epitopes for use as a heterologous, T-cell epitopecomponent of the present invention, epitope may be selected which bestmatch the MHC class I molecules present in the cell-type or cellpopulations to be targeted. Different MHC class I molecules exhibitpreferential binding to particular peptide sequences, and particularpeptide-MHC class I variant complexes are specifically recognized by theT-cell receptors (TCRs) of effector T-cells. The skilled worker can useknowledge about MHC class I molecule specificities and TCR specificitiesto optimize the selection of heterologous, T-cell epitopes used in thepresent invention.

In addition, multiple, immunogenic, T-cell epitopes for MHC class Ipresentation may be embedded in the same Shiga toxin effectorpolypeptide of the present invention, such as, e.g., for use in thetargeted delivery of a plurality of T-cell epitopes simultaneously. Anexample of a cell-targeting molecule of the present invention comprisingmultiple, CD8+ T-cell epitopes is SEQ ID NO:253.

4. Site-Specific Conjugation Sites in Shiga Toxin Effector Polypeptides

In certain embodiments of the molecules of the present invention, theShiga toxin effector polypeptide comprises a unique amino acid residue,such as, e.g., a cysteine, lysine, selenocysteine, orpyrroline-carboxy-lysine residue, which may optionally be linked, eitherdirectly or indirectly, to an agent or cargo for targeted delivery,including, e.g., a cell-targeting molecule altering agent which confersa desirable property to a cell-targeting molecule comprising the Shigatoxin effector polypeptide upon administration to a mammal (see e.g. WO2018/106895). Molecules comprising such Shiga toxin effectorpolypeptides may be equipped with a site-specific position, such as,e.g., a unique amino acid residue in the molecule, for linking othermolecules while retaining Shiga toxin function(s), such as, e.g.,stimulating cellular internalization, directing efficient intracellularrouting, and/or potent cytotoxicity. In certain further embodiments, theShiga toxin effector polypeptide is conjugated to another moiety, agent,and/or cargo, either directly or indirectly, via the unique amino acidresidue, such as, e.g., via the functional group of the unique aminoacid (see e.g. WO 2018/106895).

B. Heterologous, CD8+ T-Cell Epitope-Peptide Cargos for Delivers

The cell-targeting molecules of the present invention each comprise oneor more CD8+ T-cell epitope-peptides that are heterologous to theirrespective Shiga toxin effector polypeptide(s) and binding region(s) andwhich are not embedded or inserted within a Shiga toxin effectorpolypeptide component.

For purposes of the claimed invention, a CD8+ T-cell epitope (also knownas a MHC class I epitope or MHC class I peptide) is a molecularstructure which is comprised by an antigenic peptide and can berepresented by a linear, amino acid sequence. Commonly, CD8+ T-cellepitopes are peptides of sizes of eight to eleven amino acid residues(Townsend A, Bodmer H, Annu Rev Immunol 7: 601-24 (1989)); however,certain CD8+ T-cell epitopes have lengths that are smaller than eight orlarger than eleven amino acids long (see e.g. Livingstone A, Fathnman C,Annu Rev Immunol 5: 477-501 (1987); Green K et al., Eur J Immunol 34:2510-9 (2004)).

A CD8+ T-cell epitope is a molecular structure recognizable by an immunesystem of at least one individual, i.e. an antigenic peptide. Theheterologous, CD8+ T-cell epitope-peptide cargo of the cell-targetingmolecule of the present invention can be chosen from virtually any CD8+T-cell epitope. In certain embodiments, the heterologous, CD8+ T-cellepitope-peptide cargo has a binding affinity to a MHC class I moleculecharacterized by a dissociation constant (K_(D)) of 10′ molar or lessand/or the resulting MHC class 1-epitope-peptide complex has a bindingaffinity to a T-cell receptor (TCR) characterized by a dissociationconstant (K_(D)) of 10; molar or less.

T-cell epitopes can be empirical characterized by a binding affinity, asdescribed above. The critical structural elements of a T-cell epitopecan be identified and further analyzed using assays known to the skilledworker, such as, e.g., a combination of alanine scanning mutagenesis andbinding affinity experiments, which may also take crystallographic orother empirical structural data into account. In some instances, theenergetic contributions of some or all of the amino acid residues in theepitope-peptide to the epitope-MHC complex and/or epitope-MHC-TCRcomplex may be estimated or calculated. Certain residues/positions maybe considered or categorized as either determinant for binding affinityor neutral. Furthermore, determinant residues/positions/structures canbe further categorized as contact-determining, specificity-determining,and/or affinity determining (see e.g. Langman R, Mol Immunol 37: 555-61(2000): Greenspan N, Adv Cancer Res 80: 147-87 (2001); Cohn M, MolImmunol 42: 651-5 (2005)). Certain CD8+ T-cell epitopes may berepresented by a consensus amino acid sequence, which allows for somevariation in amino acid identity and/or positioning.

In certain embodiments of the present invention, the heterologous, CD8+T-cell epitope-peptide is at least seven amino acid residues in length.In certain embodiments of the present invention, the CD8+ T-cellepitope-peptide is bound by a TCR with a binding affinity characterizedby a K_(D) less than 10 millimolar (mM) (e.g. 1-100 μM) as calculatedusing the formula in Stone J et al., Immunology 126: 165-76 (2009).However, it should be noted that the binding affinity within a givenrange between the MHC-epitope and TCR may not correlate withantigenicity and/or immunogenicity (see e.g. Al-Ramadi B et al., JImmunol 155: 662-73 (1995)), such as due to factors like MHCI-peptide-TCR complex stability, MHC I-peptide density andMHC-independent functions of TCR cofactors such as CD8 (Baker B et al.,Imnmunity 13: 475-84 (2000); Homell T et al., J Immunol 170: 4506-14(2003); Woolridge L et al., J Immunol 171: 6650-60 (2003)).

T-cell epitopes may be chosen or derived from a number of sourcemolecules for use in the present invention. T-cell epitopes may becreated or derived from various naturally occurring proteins. T-cellepitopes may be created or derived from various naturally occurringproteins foreign to mammals, such as, e.g., proteins of microorganisms.T-cell epitopes may be created or derived from mutated human proteinsand/or human proteins aberrantly expressed by malignant human cells.T-cell epitopes may be synthetically created or derived from syntheticmolecules (see e.g., Carbone F et al., J Exp Med 167: 1767-9 (1988); DelVal M et al., J Virol 65: 3641-6 (1991); Appella E et al., Biomed PeptProteins Nucleic Acids 1: 177-84 (1995); Perez S et al., Cancer 116:2071-80 (2010)).

The CD8+ T-cell epitope-peptide of the cell-targeting molecule of thepresent invention can be chosen from various known antigens, such as,e.g., well-characterized immunogenic epitopes from human pathogens,typically the most common pathogenic viruses and bacteria.

CD8+ T-cell epitopes can be identified by reverse immunology methodsknown to the skilled worker, such as, e.g., genetic approaches, libraryscreening, and eluting peptides off of cells displaying MHC class Imolecules and sequencing them by mass-spectrometry, (see e.g. Van DerBruggen P et al., Immunol Rev 188: 51-64 (2002)).

Additionally, other MHC I-peptide binding assays based on a measure ofthe ability of a peptide to stabilize the ternary MHC-peptide complexfor a given MHC class I allele, as a comparison to known controls, havebeen developed (e.g., MHC I-peptide binding assay from ProImmune, Inc.,Sarasota, Fla., U.S.). Such approaches can help predict theeffectiveness of a putative CD8+ T-cell epitope-peptide or tocorroborate empirical evidence regarding a known CD8+ T-cell epitope.

Although any CD8+ T-cell epitope is contemplated as being used as aheterologous, CD8+ T-cell epitope of the present invention, certain CD8+T-cell epitopes may be selected based on desirable properties. Oneobjective is to create CD8+ T-cell hyper-immunized cell-targetingmolecules, meaning that the heterologous, CD8+ T-cell epitope-peptide ishighly immunogenic because it can elicit robust immune responses in vivowhen displayed complexed with a MHC class I molecule on the surface of acell.

CD8+ T-cell epitopes may be derived from a number of source moleculesalready known to be capable of eliciting a vertebrate immune response,CD8+ T-cell epitopes may be derived from various naturally occurringproteins foreign to vertebrates, such as, e.g., proteins of pathogenicmicroorganisms and non-self, cancer antigens. In particular, infectiousmicroorganisms may contain numerous proteins with known antigenic and/orimmunogenic properties. Further, infectious microorganisms may containnumerous proteins with known antigenic and/or immunogenic sub-regions orepitopes, CD8+ T-cell epitopes may be derived from mutated humanproteins and/or human proteins aberrantly expressed by malignant humancells, such as, e.g., mutated proteins expressed by cancer cells (seee.g. Sjoblom T et al., Science 314: 268-74 (2006); Wood L et al.,Science 318: 1108-13 (2007); Jones S et al., Science 321: 1801-6 (2008);Parsons D et al., Science 321: 1807-12 (2008); Wei X et al., Nat Genet43: 442-6 (2011); Govindan R et al., Cell 150: 1121-34 (2012);Vogelstein B et al., Science 339: 1546-58 (2013); Boegel S et al.,Oncoimmunology 3: e954893 (2014)).

CD8+ T-cell epitopes may be chosen or derived from a number of sourcemolecules already known to be capable of eliciting a mammalian immuneresponse, including peptides, peptide components of proteins, andpeptides derived from proteins. For example, the proteins ofintracellular pathogens with mammalian hosts are sources for CD8+ T-cellepitopes. There are numerous intracellular pathogens, such as viruses,bacteria, fungi, and single-cell eukaryotes, with well-studied antigenicproteins or peptides, CD8+ T-cell epitopes can be selected or identifiedfrom human viruses or other intracellular pathogens, such as, e.g.,bacteria like mycobacterium, fungi like toxoplasmae, and protists liketrypanosomes.

For example, there are many known immunogenic viral peptide componentsof viral proteins from viruses that infect humans. Numerous human CD8+T-cell epitopes have been mapped to peptides within proteins frominfluenza A viruses, such as peptides in the proteins HA glycoproteinsFE 17, S139/1, CH65, C05, hemagglutinin 1 (HA1), hemagglutinin 2 (HA2),nonstructural protein I and 2 (NS1 and NS 2), matrix protein 1 and 2 (M1and M2), nucleoprotein (NP), neuraminidase (NA)), and many of thesepeptides have been shown to elicit human immune responses, such as byusing ex vivo assay (see e.g. Assarsson E et al, J Virol 82: 12241-51(2008); Alexander J et al., Hum Immunol 71: 468-74 (2010); Wang M etal., PLoS One 5: e10533 (2010); Wu J et al., Clin Infect Dis 51: 1184-91(2010); Tan P et al., Human Vaccin 7: 402-9 (2011); Grant E et al.,Immunol Cell Biol 91: 184-94 (2013); Terajima M et al., Virol J 10: 244(2013)). Similarly, numerous human CD8+ T-cell epitopes have been mappedto peptide components of proteins from human cytomegaloviruses (HCMV),such as peptides in the proteins pp65 (UL83), UL128-131, immediate-early1 (IE-1; UL123), glycoprotein B, tegument proteins, and many of thesepeptides have been shown to elicit human immune responses, such as byusing ex vivo assays (Schoppel K et al., J Infect Dis 175: 533-44(1997); Elkington R et al, J Virol 77: 5226-4) (2003); Gibson L et al.,J Immunol 172: 2256-64 (2004); Ryckman B et al., J Virol 82: 60-70(2008); Sacre K et al., J Virol 82: 10143-52 (2008)).

Another example is there are many immunogenic, cancer antigens inhumans. The CD8+ T-cell epitopes of cancer and/or tumor cell antigenscan be identified by the skilled worker using techniques known in theart, such as, e.g., differential genomics, differential proteomics,immunoproteomics, prediction then validation, and genetic approacheslike reverse-genetic transfection (see e.g., Admon A et al., Mol CellProteomics 2: 388-98 (2003); Purcell A, Gorman J, Mol Cell Proteomics 3:193-208 (2004); Comber J, Philip R, Ther Adv Vaccines 2: 77-89 (2014)).There are many antigenic and/or immunogenic T-cell epitopes alreadyidentified or predicted to occur in human cancer and/or tumor cells. Forexample, T-cell epitopes have been predicted in human proteins commonlymutated or overexpressed in neoplastic cells, such as, e.g., ALK, CEA.N-acetylglucosaminyl-transferase V (GnT-V), HCA587, HER-2/neu, MAGE,Melan-A/MART-1, MUC-I, p53, and TRAG-3 (see e.g., van der Bruggen P etal., Science 254: 1643-7 (1991); Kawakami Y et al., J Exp Med 180:347-52 (1994); Fisk B et al., J Exp Med 181: 2109-17 (1995); Guilloux Yet al., J Exp Med 183: 1173 (1996); Skipper J et al., J Exp Med 183: 527(1996); Brossart P et al., 93: 4309-17 (1999); Kawashima I et al.,Cancer Res 59: 431-5 (1999); Papadopoulos K et al., Clin Cancer Res 5:2089-93 (1999); Zhu B et al., Clin Cancer Res 9: 1850-7 (2003); Li B etal., Clin Exp Immunol 140: 310-9 (2005); Ait-Tahar K et al., Int JCancer 118: 688-95 (2006); Akiyama Y et al., Cancer Immunol Immunother61: 2311-9 (2012)). In addition, synthetic variants of T-cell epitopesfrom human cancer cells have been created (see e.g., Lazoura E,Apostolopoulos V, Curr Med Chem 12: 629-39 (2005); Douat-Casassus C etal., J Med Chem 50: 1598-609 (2007)).

The term “cargo” with respect to a heterologous, CD8+ T-cell epitope isused herein to signify that the heterologous, CD8+ T-cell epitope is notembedded or inserted within a Shiga toxin effector polypeptide's Shigatoxin A1 fragment derived region or is not embedded or inserted within aShiga toxin effector polypeptide component (see e.g. WO 2015/113005).Thus, a cell-targeting molecule of the present invention may comprisemultiple, heterologous, CD8+ T-cell epitopes but only one which is acargo because the other heterologous, CD8+ T-cell epitopes are embeddedor inserted into a Shiga toxin A1 fragment region of a Shiga toxineffector polypeptide component of the cell-targeting molecule.

While any heterologous, CD8+ T-cell epitope may be used in thecompositions and methods of the present invention, certain CD8+ T-cellepitopes may be preferred based on their known and/or empiricallydetermined characteristics. Immunogenic peptide-epitopes that elicit ahuman, CD8+ T-cell responses have been described and/or can beidentified using techniques known to the skilled worker (see e.g. KalishR, J Invest Dermatol 94: 108S-111S (1990): Altman J et al., Science 274:94-6 (1996); Callan M et al., J Exp Med 187: 1395-402 (1998); Dunbar Pet al., Curr Biol 8: 413-6 (1998); Sourdive D et al., J Exp Med 188:71-82 (1998); Collins E et al., J Immunol 162: 331-7 (1999); Yee C etal., J Immunol 162: 2227-34 (1999); Burrows S et al., J Immunol 165:6229-34 (2000); Cheuk E et al., J Immunol 169: 5571-80 (2002); ElkingtonR et al, J Virol 77: 5226-(2003); Oh S et al., Cancer Res 64: 2610-8(2004); Hopkins L et al., Hum Immunol 66: 874-83 (2005); Assarsson E etal, J Virol 12241-51 (2008); Semeniuk C et al., AIDS 23: 771-7 (2009);Wang X et al., J Vis Exp 61: 3657 (2012); Song H et al., Virology 447:181-6 (2013); Chen L et al., J Virol 88: 11760-73 (2014)).

In many species, the MHC gene encodes multiple MHC-I molecular variants.Because MHC class I protein polymorphisms can affect antigen-MHC class Icomplex recognition by CD8+ T-cells, heterologous T-cell epitopes may bechosen based on knowledge about certain MHC class I polymorphisms and/orthe ability of certain antigen-MHC class I complexes to be recognized byT-cells of different genotypes.

There are well-defined peptide-epitopes that are known to beimmunogenic, MHC class I restricted, and/or matched with a specifichuman leukocyte antigen (HLA) variant(s). For applications in humans orinvolving human target cells. HLA-Class I-restricted epitopes can beselected or identified by the skilled worker using standard techniquesknown in the art. The ability of peptides to bind to human MHC Class Imolecules can be used to predict the immunogenic potential of putative,CD8+ T-cell epitopes. The ability of peptides to bind to human MHC classI molecules can be scored using software tools, CD8+ T-cell epitopes maybe chosen for use as a CD8+ heterologous, T-cell epitope component ofthe present invention based on the peptide selectivity of the HLAvariants encoded by the alleles more prevalent in certain humanpopulations. For example, the human population is polymorphic for thealpha chain of MHC class I molecules, and the variable alleles areencoded by the HLA genes. Certain T-cell epitopes may be moreefficiently presented by a specific HLA molecule, such as, e.g., thecommonly occurring HLA variants encoded by the HLA-A allele groupsHLA-A2 and HLA-A3.

When choosing CD8+ T-cell epitopes for use as a heterologous, CD8+T-cell epitope-peptide component of the cell-targeting molecule of thepresent invention, CD8+ epitopes may be selected which best match theMHC Class I molecules present in the cell-type or cell populations to betargeted. Different MHC class I molecules exhibit preferential bindingto particular peptide sequences, and particular peptide-MHC class Ivariant complexes are specifically recognized by the TCRs of effectorT-cells. The skilled worker can use knowledge about MHC class I moleculespecificities and TCR specificities to optimize the selection ofheterologous T-cell epitopes used in the present invention.

In certain embodiments of the cell-targeting molecule of the presentinvention, the heterologous, CD8+ T-cell epitope-peptide is comprisedwithin a heterologous polypeptide, such as, e.g., an antigen orantigenic protein. In certain further embodiments, the heterologouspolypeptide is no larger than 27 kDa, 28 kDa, 29 kDa, or 30 kDa.

In certain embodiments, the cell-targeting molecule of the presentinvention comprises two or more heterologous, CD8+ T-cellepitope-peptides. In certain further embodiments, the combined size ofall the heterologous, CD8+ T-cell epitope-peptides is no larger than 27kDa, 28 kDa, 29 kDa, or 30 kDa.

In certain embodiments of the cell-targeting molecule of the presentinvention, the heterologous, CD8+ T-cell epitope-peptide is processedbetter in cells with more immunoproteasomes, intermediate proteasomes,and/or thymoproteasomes as compared to standard proteasomes; however, inother embodiments the opposite is true.

When choosing CD8+ T-cell epitope-peptides for use as a heterologous,CD8+ T-cell epitope-peptide component of a cell-targeting molecule ofthe present invention, multiple factors in the MHC class I presentationsystem may be considered that can influence CD8+ T-cell epitopegeneration and transport to receptive MHC class I molecules, such as,e.g., the epitope specificity of the following factors in the targetcell: proteasome, ERAAP/ERAP1, tapasin, and TAPs can (see e.g. Akram A,Inman R. Clin Immunol 143: 99-115 (2012)).

In certain embodiments of the cell-targeting molecule of the presentinvention, the heterologous, CD8+ T-cell epitope-peptide is onlyproteolytically processed in an intact form by an intermediateproteasome (see e.g. Guillaume B et al., Proc Natl Acad Sci USA 107:18599-604 (2010); Guillaume B et al., J Immunol 189: 3538-47 (2012)).

In certain embodiments of the cell-targeting molecule of the presentinvention, the heterologous, CD8+ T-cell epitope-peptide is notdestroyed by standard proteasomes, immunoproteasomes, intermediateproteasomes, and/or thymoproteasomes, which also may depend on the celltype, cytokine environment, tissue location, etc. (see e.g., Morel S etal., Immunity 12: 107-17 (2000); Chapiro J et al., J Immunol 176:1053-61 (2006); Guillaume B et al., Proc Natl Acad Sci U.S.A. 107:18599-604 (2010); Dalet A et al., Eur J Immunol 41: 39-46 (2011); BaslerM et al., J Immunol 189: 1868-77 (2012); Guillaume B et al., J Immunol189: 3538-47 (2012)).

In certain embodiments of the cell-targeting molecule of the presentinvention, the heterologous, CD8+ T-cell epitope-peptide is considered a“weak” epitope, such as, e.g., “weak” in vivo at eliciting a CD8+ CTLresponse in a given subject or genotype group or cells derived from theaforementioned (see e.g. Cao W et al., J Immunol 157: 505-11 (1996)).

In certain embodiments of the cell-targeting molecule of the presentinvention, the heterologous, CD8+ T-cell epitope-peptide is a tumor cellepitope, such as, e.g., NY-ESO-1 157-165A (see e.g. Jager E et al. J ExpMed 187: 265-70 (1998)).

In certain embodiments of the cell-targeting molecule of the presentinvention, the heterologous, CD8+ T-cell epitope-peptide has beenmodified to have a bulky or a charged residue at its amino terminus inorder to increase ubiquitination (see e.g., Grant E et al., J Immunol155: 3750-8 (1995); Townsend A et al., J Exp Med 168: 1211-24 (1998);Kwon Y et al., Proc Natl Acad Sci U.S.A. 95: 7898-903 (1998)).

In certain embodiments of the cell-targeting molecule of the presentinvention, the heterologous, CD8+ T-cell epitope-peptide has beenmodified to have a hydrophobic amino acid residue at its carboxyterminus in order to increase proteolytic cleavage probability (seee.g., Driscoll J et al., Nature 365: 262-4 (1993); Gaczynska M et al.,Nature 365: 264-7 (1993)).

In certain embodiments of the cell-targeting molecule of the presentinvention, the heterologous, CD8+ T-cell epitope-peptide is a Tregitope.Tregitopes are functionally defined as epitope-peptides capable ofinducing an immuno-suppressive result. Examples of naturally occurringTregitopes include sub-regions of human immunoglobulin G heavy chainconstant regions (Fcs) and Fabs (see e.g., Sumida T et al., ArthritisRheum 40: 2271-3 (1997); Bluestone J, Abbas A, Nat Rev Immunol 3: 253-7(2003); Hahn B et al., J Immunol 175: 7728-37 (2005); Durinovic-Belló Iet al., Proc Natl Acad Sci USA 103: 11683-8 (2006); Sharabi A et al.,Proc Natl Acad Sci USA 103: 8810-5 (2006); De Groot A et al., Blood 112:3303-11 (2008); Sharabi A et al., J Clin Immunol 30: 34-4 (2010); MozesE, Sharabi A, Autoimmun Rev 10: 22-6 (2010)).

While the position of the heterologous, CD8+ T-cell epitope-peptide ofthe cell-targeting molecule of the present invention is not generallyrestricted. In certain embodiments of the present invention, theheterologous, CD8+ T-cell epitope-peptide is linked to thecell-targeting molecule at a location carboxy-terminal to the Shigatoxin A1 fragment derived region.

In certain embodiments, the cell-targeting molecule comprises two ormore heterologous, CD8+ T-cell epitope-peptides. In certain furtherembodiments, the multiple heterologous, CD8+ T-cell epitope-peptides arederived from different species, such as, e.g., different microorganismspecies. In certain further embodiments, the two or more, heterologous,CD8+ T-cell epitope peptides are from a bacterium and a virus or two ormore different bacterial species (see e.g. Engelhorn M et al., Mol Ther16: 773-81 (2008)). In certain further embodiments, the two or more,heterologous, CD8+ T-cell epitope peptides are from a microorganism anda human cancer cell.

C. Cell-Targeting Binding Regions

The cell-targeting molecules of the present invention comprise acell-targeting binding region capable of specifically binding anextracellular target biomolecule.

In certain embodiments, a binding region of a cell-targeting molecule ofthe present invention is a cell-targeting component, such as, e.g., adomain, molecular moiety, or agent, capable of binding specifically toan extracellular part of a target biomolecule (e.g. an extracellulartarget biomolecule) with high affinity. There are numerous types ofbinding regions known to skilled worker or which may be discovered bythe skilled worker using techniques known in the art. For example, anycell-targeting component that exhibits the requisite bindingcharacteristics described herein may be used as the binding region incertain embodiments of the cell-targeting molecules of the presentinvention.

An extracellular part of a target biomolecule refers to a portion of itsstructure exposed to the extracellular environment when the molecule isphysically coupled to a cell, such as, e.g., when the target biomoleculeis expressed at a cellular surface by the cell. In this context, exposedto the extracellular environment means that part of the targetbiomolecule is accessible by, e.g., an antibody or at least a bindingmoiety smaller than an antibody such as a single-domain antibody domain,a nanobody, a heavy-chain antibody domain derived from camelids orcartilaginous fishes, a single-chain variable fragment, or any number ofengineered alternative scaffolds to immunoglobulins (see below). Theexposure to the extracellular environment of or accessibility to a partof target biomolecule physically coupled to a cell may be empiricallydetermined by the skilled worker using methods well known in the art.

A binding region of a cell-targeting molecule of the present inventionmay be, e.g., a ligand, peptide, immunoglobulin-type binding region,monoclonal antibody, engineered antibody derivative, or engineeredalternative to antibodies.

In certain embodiments, the binding region of a cell-targeting moleculeof the present invention is a proteinaceous moiety capable of bindingspecifically to an extracellular part of target biomolecule with highaffinity. A binding region of a cell-targeting molecule of the presentinvention may comprise one or more various peptidic or polypeptidemoieties, such as randomly generated peptide sequences, naturallyoccurring ligands or derivatives thereof, immunoglobulin deriveddomains, synthetically engineered scaffolds as alternatives toimmunoglobulin domains, and the like (see e.g., WO 2005/092917; WO2007/033497; Cheung M et al., Mol Cancer 9: 28 (2010); US2013/196928; WO2014/164693; WO 2015/113005; WO 2015/113007; WO 2015/138452; WO2015/191764). In certain embodiments, a cell-targeting molecule of thepresent invention comprises a binding region comprising one or morepolypeptides capable of selectively and specifically binding anextracellular target biomolecule.

There are numerous binding regions known in the art that are useful fortargeting molecules to specific cell-types via their bindingcharacteristics, such as certain ligands, monoclonal antibodies,engineered antibody derivatives, and engineered alternatives toantibodies.

According to one specific but non-limiting aspect, the binding region ofa cell-targeting molecule of the present invention comprises a naturallyoccurring ligand or derivative thereof that retains bindingfunctionality to an extracellular target biomolecule, commonly a cellsurface receptor. For example, various cytokines, growth factors, andhormones known in the art may be used to target the cell-targetingmolecule of the present invention to the cell-surface of specificcell-types expressing a cognate cytokine receptor, growth factorreceptor, or hormone receptor. Certain non-limiting examples of ligandsinclude (alternative names are indicated in parentheses) angiogenin.B-cell activating factors (BAFFs. APRIL), colony stimulating factors(CSFs), epidermal growth factors (EGFs), fibroblast growth factors(FGFs), vascular endothelial growth factors (VEGFs), insulin-like growthfactors (IGFs), interferons, interleukins (such as IL-2, IL-6, andIL-23), nerve growth factors (NGFs), platelet derived growth factors,transforming growth factors (TGFs), and tumor necrosis factors (TNFs).

According to certain other embodiments of the cell-targeting moleculesof the present invention, the binding region comprises a syntheticligand capable of binding an extracellular target biomolecule (see e.g.Liang S et al., J Mol Med 84: 764-73 (2006); Ahmed S et al., Anal Chem82: 7533-41 (2010); Kaur K et al., Methods Mol Biol 1248: 239-47(2015)).

In certain embodiments, the binding region comprises a peptidomimetic,such as, e.g., an AApeptide, gamma-AApeptide, and/or sulfono-γ-AApeptide(see e.g., Pilsl L, Reiser O, Amino Acids 41: 709-18 (2011); Akram O etal., Mol Cancer Res 12: 967-78 (2014); Wu H et al., Chemistry 21: 2501-7(2015); Teng P et al., Chemistry 2016 Mar. 4)).

According to one specific, but non-limiting aspect, the binding regionmay comprise an immunoglobulin-type binding region. The term“immunoglobulin-type binding region” as used herein refers to apolypeptide region capable of binding one or more target biomolecules,such as an antigen or epitope. Binding regions may be functionallydefined by their ability to bind to target molecules.Immunoglobulin-type binding regions are commonly derived from antibodyor antibody-like structures; however, alternative scaffolds from othersources are contemplated within the scope of the term.

Immunoglobulin (Ig) proteins have a structural domain known as an Igdomain. Ig domains range in length from about 70-110 amino acid residuesand possess a characteristic Ig-fold, in which typically 7 to 9antiparallel beta strands arrange into two beta sheets which form asandwich-like structure. The Ig fold is stabilized by hydrophobic aminoacid interactions on inner surfaces of the sandwich and highly conserveddisulfide bonds between cysteine residues in the strands. Ig domains maybe variable (IgV or V-set), constant (IgC or C-set) or intermediate (IgIor I-set). Some Ig domains may be associated with a complementaritydetermining region (CDR), also called a “complementary determiningregion,” which is important for the specificity of antibodies binding totheir epitopes. Ig-like domains are also found in non-immunoglobulinproteins and are classified on that basis as members of the Igsuperfamily of proteins. The HUGO Gene Nomenclature Committee (HGNC)provides a list of members of the Ig-like domain containing family.

An immunoglobulin-type binding region may be a polypeptide sequence ofan antibody or antigen-binding fragment thereof wherein the amino acidsequence has been varied from that of a native antibody or an Ig-likedomain of a non-immunoglobulin protein, for example by molecularengineering or selection by library screening. Because of the relevanceof recombinant DNA techniques and in vitro library screening in thegeneration of immunoglobulin-type binding regions, antibodies can beredesigned to obtain desired characteristics, such as smaller size, cellentry, or other improvements for in vivo and/or therapeuticapplications. The possible variations are many and may range from thechanging of just one amino acid to the complete redesign of; forexample, a variable region. Typically, changes in the variable regionwill be made in order to improve the antigen-binding characteristics,improve variable region stability, or reduce the potential forimmunogenic responses.

There are numerous immunoglobulin-type binding regions contemplated ascomponents of the present invention. In certain embodiments, theimmunoglobulin-type binding region is derived from an immunoglobulinbinding region, such as an antibody paratope capable of binding anextracellular target biomolecule. In certain other embodiments, theimmunoglobulin-type binding region comprises an engineered polypeptidenot derived from any immunoglobulin domain but which functions like animmunoglobulin binding region by providing high-affinity binding to anextracellular target biomolecule. This engineered polypeptide mayoptionally include polypeptide scaffolds comprising or consistingessentially of complementary determining regions from immunoglobulins asdescribed herein.

There are also numerous binding regions in the prior art that are usefulfor targeting polypeptides to specific cell-types via theirhigh-affinity binding characteristics. In certain embodiments of thecell-targeting molecules of the present invention, the binding regioncomprises immunoglobulin domain selected from the group which includesautonomous V_(H) domains, single-domain antibody domains (sdAbs),heavy-chain antibody domains derived from camelids (V_(H)H fragments orV_(H) domain fragments), heavy-chain antibody domains derived fromcamelid V_(H)H fragments or V_(H) domain fragments, heavy-chain antibodydomains derived from cartilaginous fishes, immunoglobulin new antigenreceptors (IgNARs), V_(NAR) fragments, single-chain variable (scFv)fragments, nanobodies, Fd fragments consisting of the heavy chain andCHI domains, antibody variable domain (Fv) fragments, permutated Fvs(pFv), single chain Fv-C_(H)3 minibodies, dimeric C_(H)2 domainfragments (C_(H)2D), Fc antigen binding domains (Fcabs), isolatedcomplementary determining region 3 (CDR3) fragments, constrainedframework region 3, CDR3, framework region 4 (FR3-CDR3-FR4)polypeptides, small modular immunopharmaceutical (SMIP) domains, scFv-Fcfusions, one-arm single-chain Fab constructs, multimerizing scFvfragments (diabodies, triabodies, tetrabodies), disulfide-stabilizedantibody variable (Fv) fragments, disulfide-stabilized antigen-binding(Fab) fragments consisting of the V_(L), V_(H), C_(L) and C_(H)1domains, bivalent nanobodies, bivalent minibodies, bivalent F(ab′)₂fragments (Fab dimers), bispecific tandem V_(H)H fragments, bispecifictandem scFv fragments, bispecific nanobodies, bispecific minibodies,one-arm single-chain Fab heterodimeric bispecific constructs, and anygenetically manipulated counterparts of the foregoing that retains itsbinding functionality (Brinkmann U et al., J Mol Biol 268: 107-17(1997); W6m A, Plückthun A, J Mol Biol 305: 989-1010 (2001); Xu L etal., Chem Biol 9: 933-42 (2002); Wikman M et al., Protein Eng Des Sel17: 455-62 (2004); Binz H et al., Nat Biotechnol 23: 1257-68 (2005); HeyT et al., Trends Biotechnol 23:514-522 (2005); Holliger P, Hudson P, NatBiotechnol 23: 1126-36 (2005); Gill D, Damle N, Curr Opin Biotech 17:653-8 (2006); Koide A, Koide S, Methods Mol Biol 352: 95-109 (2007);Byla P et al., J Biol Chem 285: 12096 (2010); Zoller F et al., Molecules16: 2467-85 (2011); Alfarano P et al., Protein Sci 21: 1298-314 (2012);Madhurantakam C et al., Protein Sci 21: 1015-28 (2012); Varadamsetty Get al., J Mol Bol 424: 68-87 (2012); Reichen C et al., J Struct Biol185: 147-62 (2014); Schanzer J et al., J Biol Chem 289: 18693-706(2014)).

In certain embodiments, the binding region of the cell-targetingmolecule of the present invention is selected from the group whichincludes autonomous V_(H)domains (such as, e.g., from camelids, murine,or human sources), single-domain antibody domains (sdAbs), heavy-chainantibody domains derived from camelids (V_(H)H fragments or V_(H) domainfragments), heavy-chain antibody domains derived from camelid V_(H)Hfragments or V_(H) domain fragments, heavy-chain antibody domainsderived from cartilaginous fishes, immunoglobulin new antigen receptors(IgNARs), V_(NAR) fragments, single-chain variable (scFv) fragments,nanobodies, “camelized” or “camelised” scaffolds comprising a V_(H)domain, Fd fragments consisting of the heavy chain and CHI domains,single chain Fv-C_(H)3 minibodies, dimeric C_(H)2 domain fragments(C_(H)2D), Fc antigen binding domains (Fcabs), isolated complementarydetermining region 3 (CDR3) fragments, constrained framework region 3,CDR3, framework region 4 (FR3-CDR3-FR4) polypeptides, small modularimmunopharmaceutical (SMIP) domains, scFv-Fc fusions, multimerizing scFvfragments (diabodies, triabodies, tetrabodies), disulfide-stabilizedantibody variable (Fv) fragments (dsFv), disulfide-stabilizedantigen-binding (Fab) fragments consisting of the V_(L), V_(H), C_(L)and C_(H)1 domains, single-chain variable-region fragments comprising adisulfide-stabilized heavy and light chain (sc-dsFvs), bivalentnanobodies, bivalent minibodies, bivalent F(ab′)₂ fragments (Fabdimers), bispecific tandem V_(H)H fragments, bispecific tandem scFvfragments, bispecific nanobodies, bispecific minibodies, and anygenetically manipulated counterparts of the foregoing that retain itsparatope and binding function (see Ward E et al., Nature 341: 544-6(1989); Davies J, Riechmann L, Biotechnology (NY) 13: 475-9 (1995);Reiter Y et al. Mol Biol 290: 685-98 (1999); Riechmann L, Muyldermans S,J Immunol Methods 231: 25-38 (1999); Tanha J et al., J Immunol Methods263: 97-109 (2002); Vranken W et al., Biochemistry 41: 8570-9 (2002):Dottorini T et al., Biochemistry 43: 622-8 (2004); Jespers L et al., JMol Biol 337: 893-903 (2004); Jespers L et al., Nat Biotechnol 22:1161-5 (2004); Spinelli S et al., FEBS Lett 564: 35-40 (2004); To R etal., J Biol Chem 280: 41395-403 (2005); Tanha J et al., Protein Eng DesSel 19: 503-9 (2006); Saerens D et al., Curr Opin Pharmacol 8: 600-8(2008); Dimitrov D, MAbs 1: 26-8 (2009); Chen I et al., Mol Biosyst 6:1307-15 (2010); Huang Y et al., J Biol Chem 285: 7880-91 (2010); Lee etal., Biochem Biophys Res Commun 411: 348-53 (2011); Ahmad Z et al., ClinDev Immunol 2012: 980250 (2012); Baral T et al., PLoS One 7: e30149(2012); Weiner L, Cell 148: 1081-4 (2012)).

In certain embodiments, the binding region of a molecule of the presentinvention is multivalent and bispecific but the specificity is to asingle target, i.e. the bispecificity is due to different bindingregions binding different extracellular epitopes present on the same ora single extracellular target biomolecule (see e.g. Schanzer J et al.,Antimicrob Agents Chemother 55: 2369-78 (2011)).

Immunoglobulin domains and/or fragments may be modified for use as acell-targeting moiety in a cell-targeting molecule of the presentinvention by the addition or removal of a cysteine residue(s) and/ordisulfide bond(s) and/or modification of other residues (see e.g. YoungN et al., FEBS Lett 377: 135-9 (1995); Wirtz P. Steipe B, Protein Sci 8:2245-50 (1999); Barthelemy P et al., J Biol Chem 283: 3639-54 (2008);Arabi-Ghahroudi M et al., Protein Eng Des Sel 22: 59-66 (2009); Zhao Jet al., Int J Mol Sci 12: 1-11 (2010); Duan Y et al., Mol Immunol 51:188-96 (2012); Kim D et al., Protein Eng Des Sel 25: 581-9 (2012) Gil D.Schrum A. Adv Biosci Biotechnol 4: 73-84 (2013)).

In certain embodiments, the cell-targeting molecule of the presentinvention comprises an immunoglobulin-type binding region whichcomprises an immunoglobulin domain and/or Ig-fold structure having anintra-domain disulfide bond, such as, e.g., the disulfide bond foundnatively between the B and F β strands of certain immunoglobulins and/ora disulfide bond between their heavy and light chains of or derived froman immunoglobulin. However, in certain embodiments of the cell-targetingmolecule of the present invention, the molecules are very stable eventhough they do not comprise an intra-domain disulfide bond or anyintra-domain disulfide bond within one or more immunoglobulin-typebinding regions (see e.g. Proba K et al., Biochemistry 37: 13120-7(1998); Wmrn A. Plückthun A, Biochemistry 37: 13120-7 (1998); Wöm A.Plückthun A, FEBS Lett 427: 357-61 (1998); Ramm K et al., J Mol Biol290: 535-46 (1999); Tanaka T, Rabbitts T, J Mol Biol 376: 749-57(2008)).

In certain embodiments, cell-targeting molecule of the present inventioncomprises an immunoglobulin-type binding region derived from animmunoglobulin which has been engineered with certain camelid V_(H)H“tetrad” mutations to improve solubility, to improve stability, and/orotherwise “camelize” the binding region (see e.g. Vincke C et al., JBiol Chem 284: 3273-84 (2009); Perchiacca J et al., Proteins 79: 2637-47(2011); Gil D, Schrum A, Adv Biosci Biotechnol 4: 73-84 (2013)).

The skilled worker may use numerous approaches known in the art tominimize and/or prevent aggregation of molecules comprisingimmunoglobulin domains and/or fragments or otherwise comprisingcomponents derived from immunoglobulins (see e.g. Jespers L et al., NatBiotechnol 22: 1161-5 (2004): Daugherty A, Mrsny R, Adv Drug Deliv Rev58: 686-706 (2006); Christ D et al., Protein Eng Des Sel 20: 413-6(2007); Wang W et al., J Pharma Sci 96: 1-26 (2007); Famm K et al., JMol Biol 376: 926-31 (2008); Arabi-Ghahroudi M et al., Protein Eng DesSel 22: 59-66 (2009); Wang X et al., MAbs 1: 254-267 (2009): Dudgeon Ket al., Proc Natl Acad Sci USA 109: 10879-84 (2012); Kim D et al.,Methods Mol Biol 911: 355-72 (2012); Perchiacca J et al., Protein EngDes Sel 25: 591-601 (2012); Schaefer J, Plückthun A, J Mol Biol 417:309-335 (2012); Buchanan A et al., MAbs 5: 255-62 (2013); Lee C et al.,Trends Biotechnol 31: 612-(2013); Tiller T et al., MAbs 5: 445-470(2013); Kim D et al., Biochim Biophys Acta 1844: 1983-2001 (2014);Perchiacca J et al., Protein Eng Des Sel 27: 29-39 (2014); Rouet R etal., FEBS Lett 588: 269-77 (2014); Swift J et al., Protein Eng Des Sel27: 405-9 (2014); Enever C et al., Protein Eng Des Sel 28: 59-68(2015)).

The skilled worker may use the addition or maintenance of intermoleculardisulfide bonds to stabilize certain binding regions of thecell-targeting molecules of the present invention (see e.g. GlockshuberR et al., Biochemistry 29: 1362-7 (1990): Stanfield R et al., Science305: 1770-3 (2004); Hagihara Y et al., J Biol Chem 282: 36489-95 (2007);Chan P et al., Biochemistry 47: 11041-54 (2008); Saerens D et al., J MolBiol 478-88 (2008); Hussack G et al., PLoS One 6: e28218 (2011); GovaertJ et al., J Biol Chem 287: 1970-9 (2012); Kim D et al., Protein Eng DesSel 25: 581-9 (2012); Gil D, Schrum A, Adv Biosci Biotechnol 4: 73-84(2013); McConnell A et al., Protein Eng Des Sel 25: 581-9 (2013); FeigeM et al., Proc Natl Acad Sci USA 111: 8155-60 (2014); Hagihara Y,Saerens D, Biochim Biophys Acta 1844: 2016-2023 (2014); Kim D et al.,Mabs 6: 219-35 (2014)).

For example, in certain embodiments, the cell-targeting molecule of thepresent invention is engineered to minimize the formation of unwanted,intermolecular associations, multimers, and/or aggregates, such as,e.g., by using disulfide-stabilized scFvs, Fv fragments, or Fabs (seee.g. Reiter Y et al., J Biol Chem 269: 18327-31 (1994); Kuan C, PastanI, Biochemistry 35: 2872-7 (1996); Almog O et al., Proteins 31: 128-38(1998); Schoonjans R et al., J Immunol 165: 7050-7 (2000); Olafsen T etal., Protein Eng Des Sel 17: 21-7 (2004); Gil D, Schrum A, Adv BiosciBiotechnol 4: 73-84 (2013); U.S. 20120283418): base loop connections(see e.g. Brinkmann U et al., J Mol Biol 268: 107-17 (1997)); and/orother modifications, such as the addition of charged resides, glycans,and/or immunoglobulin-domain truncations (see e.g. Gong R et al., MolPharm 10: 2642-52 (2013): Lee C et al., Trends Biotechnol 31: 612-20(2013); Goldman E et al., Protein Expr Purif 95: 226-32 (2014)).

In certain embodiments of the present invention, the cell-targetingmolecule of the present invention comprises an immunoglobulin-typebinding region which is an scFv engineered not to aggregate, such as,e.g., by using a shorter linker (typically less than twelve amino acidresidues) and/or disulfide-stabilized linker that links the heavy andlight chain regions of the scFv (see e.g., Brinkmann U et al., Proc NatlAcad Sci USA 90: 7538-42 (1993): Whitlow M et al., Protein Engineering6: 989-95 (1993); Reiter Y et al., Biochemistry 33: 5451-9 (1994); GongR et al., Molecular Pharmaceutics 10: 2642-52 (2013)).

There are a variety of binding regions comprising polypeptides derivedfrom the constant regions of immunoglobulins which may be used a bindingregion(s) of a cell-targeting molecule of the present invention, suchas, e.g., engineered dimeric Fc domains, monomeric Fcs (mFcs), scFv-Fcs,V_(H)H-Fcs, C_(H)2 domains, monomeric C_(H)3s domains (mC_(H)3s),synthetically reprogrammed immunoglobulin domains, and/or hybrid fusionsof immunoglobulin domains with ligands (Hofer T et al., Proc Natl AcadSci USA 105: 12451-6 (2008): Xiao J et al., J Am Chem Soc 131: 13616-8(2009); Xiao X et al., Biochem Biophys Res Commun 387: 387-92 (2009);Wozniak-Knopp G et al., Protein Eng Des Sel 23 289-97 (2010); Gong R etal., PLoS ONE 7: e42288 (2012): Wozniak-Knopp G et al., PLoS ONE 7:e30083 (2012); Ying T et al., J Biol Chem 287: 19399-408 (2012): Ying Tet al., J Biol Chem 288: 25154-64 (2013); Chiang M et al., J Am Chem Soc136: 3370-3 (2014); Rader C, Trends Biotechnol 32: 186-97 (2014); Ying Tet al., Biochimica Biophys Acta 1844: 1977-82 (2014)).

In accordance with certain other embodiments, the binding regioncomprises an immunoglobulin domain(s) that is not from a traditionalimmunoglobulin but rather is from a cell-membrane bound receptor whichfunctions as part of the immune system. In certain embodiments, thecell-targeting binding region of the cell-targeting molecule of thepresent invention comprises or consists essentially of a single-chainT-cell receptor variable fragment (scTv), a single-chain TCR (scTCR),disulfide-stabilized T-cell receptor variable fragment (dsTv), and/orT-cell receptor variable fragment disulfide-stabilized Fv heterodimer(TCR dsFv heterodimer).

In certain embodiments, the cell-targeting binding region of thecell-targeting molecule of the present invention is a soluble,single-chain T-cell receptor variable fragment (soluble scTv) and/or TCRdsFv heterodimer (see e.g. Novotny J et al., Proc Natl Acad Sci USA 88:8646-50 (1991); Soo Hoo, W et al., Proc Natl Acad Sci USA 89: 4759-63(1992); Shusta E et al., J Mol Biol 292: 949-56 (1999); Holler P et al.,Proc Natl Acad Sci USA 97: 5387-92 (2000); Boulter J et al., Protein Eng16: 50 707-11 (2003); Li Y et al., Nat Biotechnol 23: 349-54 (2005);Weber K et al., Proc Natl Acad Sci USA 102: 19033-8 (2005); Dunn S etal., Protein Sci 15: 710-21 (2006); Richman S, Kranz D, Biomol Eng 24:361-73 (2007); Varela-Rohena A et al., Nat Med 14: 1390-5 (2008);Sadelain M et al., Curr Opin Immunol 21: 215-23 (2009); Aggen D et al.,Protein Eng Des Sel 24: 361-72 (2011); WO1999060120; WO2001057211:WO2003020763: U.S. Pat. No. 7,329,731). Unlike most immunoglobulins,naturally occurring scTvs typically bind with moderate affinity to anepitope-peptide-MHC protein complex. While scTvs in isolation bind tosuch cell-surface targets (i.e. extracellular target biomoleculesphysically coupled to cells in the form of pMHCs), scTvs also can retaintheir binding specificity for cell-targeting upon fusion to an effectormolecule, such as a toxin protein (see e.g. Epel M et al., CancerImmunol Immunother 51: 563-73 (2002)). While such scTv's can beengineered to recognize new targets with high-affinity binding,specificity and selectivity, the targets are typically MHCprotein-non-self epitope-peptide complexes which are displayed on thesurfaces of vertebrate cells. However, naturally occurring TCRs whichhave been deleted during thymic selection often bind self-epitope-MHCcomplexes with high affinities. Furthermore, the scTv may be mutated inits variable domain(s) to improve the affinity and/or stability ofdesired binding interactions (see e.g. Shusta E et al., Nat Bioteclmol18: 754-9 (2000): Richman S et al., Mol Immunol 46: 902-16 (2009)). Theintroduction of solubility increasing mutations in a scTCR and/or anon-native disulfide bond in the TCR invariant region, to make dsTCRs,can greatly increase the stability and folding characteristics of a scTv(see e.g. Molloy P et al., Curr Opin Pharmacol 5: 438-43 (2005);WO2003020763). In addition, it may improve stability and production of ascTv by orienting the domains of the scTCR in the amino-to-carboxyorientation of Va domain-linker-VD3 domain (Loset G et al., Protein EngDes Sel 20: 461-72 (2007): Richman S et al., Mol Immunol 46: 902-16(2009)). The introduction of various mutations may also improveexpression in a host cell system, e.g. in yeast cells (see e.g. RichmanS et al., Mol Immunol 46: 902-16 (2009)).

In accordance with certain other embodiments, the binding regioncomprises an engineered, alternative scaffold to immunoglobulin domains.Engineered alternative scaffolds are known in the art which exhibitsimilar functional characteristics to immunoglobulin-derived structures,such as high-affinity and specific binding of target biomolecules, andmay provide improved characteristics to certain immunoglobulin domains,such as, e.g., greater stability or reduced immunogenicity. Generally,alternative scaffolds to immunoglobulins are less than kilodaltons(kDa), consist of a single polypeptide chain, lack cysteine residues,and exhibit relatively high thermodynamic stability.

In certain embodiments of the cell-targeting molecules of the presentinvention, the immunoglobulin-type binding region is selected from thegroup which includes engineered, Armadillo repeat polypeptides (ArmRPs);engineered, fibronectin-derived, 10^(th) fibronectin type III (10Fn3)domains (monobodies, AdNectins™, or AdNexinsm); engineered,tenascin-derived, tenascin type III domains (Centryns™); engineered,ankyrin repeat motif containing polypeptides (DARPins™); engineered,low-density-lipoprotein-receptor-derived, A domains (LDLR-A) (Avimers™);lipocalins (anticalins); engineered, protease inhibitor-derived, Kunitzdomains; engineered, Protein-A-derived, Z domains (Affibodies™):engineered, gamma-B crystallin-derived scaffold or engineered,ubiquitin-derived scaffolds (Affilins); Sac7d-derived polypeptides(Nanoffitins® or affitins); engineered, Fyn-derived, SH2 domains(Fynomers®); and engineered antibody mimics and any geneticallymanipulated counterparts of the foregoing that retains its bindingfunctionality (Wörn A, Plickthun A. J Mol Biol 305: 989-1010 (2001); XuL et al., Chem Biol 9: 933-42 (2002); Wikman M et al., Protein Eng DesSel 17: 455-62 (2004); Binz H et al., Nat Biotechnol 23: 1257-68 (2005):Hey T et al., Trends Biotechnol 23:514-522 (2005); Holliger P. Hudson P,Nat Biotechnol 23: 1126-36 (2005): Gill D, Damle N. Curr Opin Biotech17: 653-8 (2006); Koide A, Koide S, Methods Mol Biol 352: 95-109 (2007);Byla P et al., J Biol Chem 285: 12096 (2010); Zoller F et al., Molecules16: 2467-85 (2011): Alfarano P et al., Protein Sci 21: 1298-314 (2012);Madhurantakam C et al., Protein Sci 21: 1015-28 (2012); Varadansetty Get al., J Mol Biol 424: 68-87 (2012)).

For example, there is an engineered Fn3(CD20) binding region scaffoldwhich exhibits high-affinity binding to CD20 expressing cells (NatarajanA et al., Clin Cancer Res 19: 6820-9 (2013)).

For example, numerous alternative scaffolds have been identified whichbind to the extracellular receptor HER2 (see e.g. Wikman M et al.,Protein Eng Des Sel 17: 455-62 (2004); Orlova A et al. Cancer Res 66:4339-8 (2006); Ahlgren S et al., Bioconjug Chem 19: 235-43 (2008);Feldwisch J et al., J Mol Biol 398: 232-47 (2010): U.S. Pat. Nos.5,578,482; 5,856,110; 5,869,445; 5,985,553; 6,333,169; 6,987,088;7,019,017; 7,282,365; 7,306,801; 7,435,797; 7,446,185; 7,449,480;7,560,111; 7,674,460; 7,815,906; 7,879,325; 7,884,194; 7,993,650;8,241,630; 8,349,585; 8,389,227; 8,501,909; 8,512,967; 8,652,474; andU.S. patent application 20110059090). In addition to alternativeantibody formats, antibody-like binding abilities may be conferred bynon-proteinaceous compounds, such as, e.g., oligomers. RNA molecules,DNA molecules, carbohydrates, and glycocalyxcalixarenes (see e.g.Sansone F, Casnati A, Chem Soc Rev 42: 4623-39 (2013)) or partiallyproteinaceous compounds, such as, e.g., phenol-formaldehyde cyclicoligomers coupled with peptides and calixarene-peptide compositions (seee.g. U.S. Pat. No. 5,770,380).

In certain embodiments, it may be preferable to use protease-resistantimmunoglobulin domains and/or synthetically stabilized scFv fragments,such as to avoid instability during storage or after administration butbefore reaching a target cell (see e.g. Ewert S et al., Methods 34:184-99 (2004); Honegger et al., Protein Eng Des Sel 22: 135-47 (2009);Miller et al., Protein Eng Des Sel 23: 549-57 (2010); Hussack G et al.,Protein Eng Des Sel 27: 191-8 (2014)).

Any of the above binding region structures may be used as a component ofa cell-targeting molecule of the present invention as long as thebinding region component has a dissociation constant of 10⁻⁵ to 10⁻¹²moles per liter, preferably less than 200 nanomolar (nM), towards anextracellular target biomolecule.

In certain embodiments, the cell-targeting molecules of the presentinvention comprise a Shiga toxin effector polypeptide of the presentinvention linked and/or fused to a binding region capable ofspecifically binding an extracellular part of a target biomolecule or anextracellular target biomolecule. Extracellular target biomolecules maybe selected based on numerous criteria, such as a criterion describedherein.

Extracellular Target Biomolecules Bound by the Binding Regions

In certain embodiments, the binding region of a cell-targeting moleculesof the present invention comprises a proteinaceous region capable ofbinding specifically to an extracellular part of a target biomolecule oran extracellular target biomolecule, preferably which is physicallycoupled to the surface of a cell-type of interest, such as, e.g., acancer cell, tumor cell, plasma cell, infected cell, or host cellharboring an intracellular pathogen. Preferably, the targeted cell-typewill be expressing a MHC class I molecule(s). Target biomolecules boundby the binding region of a cell-targeting molecule of the presentinvention may include biomarkers over-proportionately or exclusivelypresent on cancer cells, immune cells, and/or cells infected withintracellular pathogens, such as, e.g., viruses, bacteria, fungi,prions, or protozoans.

The term “target biomolecule” refers to a biological molecule, commonlya proteinaceous molecule or a protein modified by post-translationalmodifications, such as glycosylation, that is bound by a binding regionof a cell-targeting molecule of the present invention resulting in thetargeting of the cell-targeting molecule to a specific cell, cell-type,and/or location within a multicellular organism.

For purposes of the present invention, the term “extracellular” withregard to a target biomolecule refers to a biomolecule that has at leasta portion of its structure exposed to the extracellular environment. Theexposure to the extracellular environment of or accessibility to a partof target biomolecule coupled to a cell may be empirically determined bythe skilled worker using methods well known in the art. Non-limitingexamples of extracellular target biomolecules include cell membranecomponents, transmembrane spanning proteins, cell membrane-anchoredbiomolecules, cell-surface-bound biomolecules, and secretedbiomolecules.

With regard to the present invention, the phrase “physically coupled”when used to describe a target biomolecule means covalent and/ornon-covalent intermolecular interactions couple the target biomolecule,or a portion thereof, to the outside of a cell, such as a plurality ofnon-covalent interactions between the target biomolecule and the cellwhere the energy of each single interaction is on the order of at leastabout 1-5 kiloCalories (e.g., electrostatic bonds, hydrogen bonds, ionicbonds, Van der Walls interactions, hydrophobic forces, etc.). Allintegral membrane proteins can be found physically coupled to a cellmembrane, as well as peripheral membrane proteins. For example, anextracellular target biomolecule might comprise a transmembrane spanningregion, a lipid anchor, a glycolipid anchor, and/or be non-covalentlyassociated (e.g. via non-specific hydrophobic interactions and/or lipidbinding interactions) with a factor comprising any one of the foregoing.

Extracellular parts of target biomolecules may include various epitopes,including unmodified polypeptides, polypeptides modified by the additionof biochemical functional groups, and glycolipids (see e.g. U.S. Pat.No. 5,091,178: EP2431743).

The binding regions of the cell-targeting molecules of the presentinvention may be designed or selected based on numerous criteria, suchas the cell-type specific expression of their target biomolecules, thephysical localization of their target biomolecules with regard tospecific cell-types, and/or the properties of their target biomolecules.For example, certain cell-targeting molecules of the present inventioncomprise binding regions capable of binding cell-surface targetbiomolecules that are expressed at a cellular surface exclusively byonly one cell-type of a species or only one cell-type within amulticellular organism. It is desirable, but not necessary, that anextracellular target biomolecule be intrinsically internalized or bereadily forced to internalize upon interacting with a cell-targetingmolecule of the present invention.

It will be appreciated by the skilled worker that any desired targetbiomolecule may be used to design or select a suitable binding region tobe associated and/or coupled with a Shiga toxin effector polypeptide toproduce a cell-targeting molecule of the present invention.

The general structure of the cell-targeting molecules of the presentinvention is modular, in that various, diverse cell-targeting bindingregions may be used with various Shiga toxin effector polypeptides andCD8+ T-cell epitope-peptides to provide for diverse targeting anddelivery of various epitopes to the MHC class I system of diverse targetcell-types. Optionally, a cell-targeting molecule of the invention (e.g.protein) may further comprise a carboxy-terminal endoplasmicretention/retrieval signal motif, such as, e.g., the amino acids KDEL atthe carboxy terminus of a proteinaceous component of the cell-targetingmolecule (see e.g. PCT/US2015/19684).

D. Linkages Connecting Components of the Cell-Targeting Molecules of theInvention

Individual cell-targeting binding regions, Shiga toxin effectorpolypeptides, CD8+ T-cell epitope cargos, and/or other components of thecell-targeting molecules present invention may be suitably linked toeach other via one or more linkers well known in the art and/ordescribed herein (see e.g., WO 2014/164693; WO 2015/113005: WO2015/113007; WO 2015/138452: WO 2015/191764). Individual polypeptidesubcomponents of the binding regions, e.g. heavy chain variable regions(V_(H)), light chain variable regions (V_(L)), CDR, and/or ABR regions,may be suitably linked to each other via one or more linkers well knownin the art and/or described herein. Proteinaceous components of theinvention, e.g., multi-chain binding regions, may be suitably linked toeach other or other polypeptide components of the invention via one ormore linkers well known in the art. Peptide components of the invention,e.g., a heterologous, CD8+ T-cell epitope-peptide cargos, may besuitably linked to another component of the invention via one or morelinkers, such as a proteinaceous linker, which is well known in the art.

Suitable linkers are generally those which allow each polypeptidecomponent of the present invention to fold with a three-dimensionalstructure very similar to the polypeptide components producedindividually without any linker or another component associated with it.Suitable linkers include single amino acids, peptides, polypeptides, andlinkers lacking any of the aforementioned, such as variousnon-proteinaceous carbon chains, whether branched or cyclic (see e.g.Alley S et al., Bioconjug Chem 19: 759-65 (2008); Ducry L, Stump B.Bioconjug Chem 21: 5-13 (2010)).

Suitable linkers may be proteinaceous and comprise one or more aminoacids, peptides, and/or polypeptides. Proteinaceous linkers are suitablefor both recombinant fusion proteins and chemically linked conjugates. Aproteinaceous linker typically has from about 2 to about 50 amino acidresidues, such as, e.g., from about 5 to about 30 or from about 6 toabout 25 amino acid residues. The length of the linker selected willdepend upon a variety of factors, such as, e.g., the desired property orproperties for which the linker is being selected. In certainembodiments, the linker is proteinaceous and is linked near the terminusof a protein component of the present invention, typically within about20 amino acids of the terminus. Frequently, suitable proteinaceouslinkers comprise stretches of glycines and/or serines for flexibilitycombined with one or more charged residues, such as, e.g., a glutamateand/or lysine residue(s) for solubility (see e.g. Whitlow M et al.,Protein Engineering 6: 989-95 (1993)).

Suitable linkers may be non-proteinaceous, such as, e.g. chemicallinkers.

Suitable methods for linkage of the components of the cell-targetingmolecules of the present invention may be by any method presently knownin the art for accomplishing such, as long as the attachment does notsubstantially impede the binding capability of the cell-targetingbinding region and/or when appropriate the desired Shiga toxin effectorfunction(s) as measured by an appropriate assay, including assaysdescribed herein. For example, disulfide bonds and thioether bonds maybe used to link two or more proteinaceous components of a cell-targetingmolecule of the present invention.

For the purposes of the cell-targeting molecules of the presentinvention, the specific order or orientation is not fixed for thecomponents unless stipulated. The arrangement of the Shiga toxineffector polypeptide(s), heterologous, CD8+ T-cell epitope cargo(s), thebinding region(s), and any optional linker(s), in relation to each otheror the entire cell-targeting molecule is not fixed (see e.g. FIG. 1)unless specifically noted. In general, the components of thecell-targeting molecules of the present invention may be arranged in anyorder provided that the desired activity(ies) of the binding region,Shiga toxin effector polypeptide, and heterologous, CD8+ T-cell epitopeare not eliminated.

II. Examples of Specific Structural Variations of the Cell-TargetingMolecules of the Present Invention

The cell-targeting molecules of the present invention comprise a Shigatoxin A Subunit effector polypeptide, a cell-targeting binding region,and a heterologous, CD8+ T-cell epitope-peptide cargo which is notembedded or inserted in the Shiga toxin A1 fragment region and/or theShiga toxin A Subunit effector polypeptide. A cell-targeting moleculewith the ability to deliver a CD8+ T-cell epitope cargo to the MHC classI presentation pathway of a target cell may be created, in principle, bylinking any heterologous, CD8+ T-cell epitope-peptide to any combinationof cell-targeting binding region and Shiga toxin A Subunit effectorpolypeptide as long as the resulting cell-targeting molecule has acellular internalization capability (such as, e.g., via endocytosis)provided by at least the Shiga toxin effector, the cell-targetingmoiety, or the structural combination of them together, and as long asthe Shiga toxin effector polypeptide component or the cell-targetingmolecule structure as a whole, provides, once inside a target cell,sufficient subcellular routing to a subcellular compartment competentfor delivery of the T-cell epitope-peptide to the MHC class Ipresentation pathway of the target cell, such as, e.g., to the cytosolor the endoplasmic reticulum (ER).

The cell-targeting molecules of the present invention each comprise atleast one Shiga toxin A Subunit effector polypeptide derived from atleast one A Subunit of a member of the Shiga toxin family. In certainembodiments, the Shiga toxin effector polypeptide of the cell-targetingmolecule of the present invention comprises or consists essentially of atruncated Shiga toxin A Subunit. Truncations of Shiga toxin A Subunitsmight result in the deletion of an entire epitope(s) and/or epitoperegion(s), B-cell epitopes, CD4+ T-cell epitopes, and/or furin-cleavagesites without affecting Shiga toxin effector functions, such as, e.g.,catalytic activity and cytotoxicity. The smallest Shiga toxin A Subunitfragment shown to exhibit full enzymatic activity was a polypeptidecomposed of residues 1-239 of Slt1A (LaPointe P et al., J Biol Chem 280:23310-18 (2005)). The smallest Shiga toxin A Subunit fragment shown toexhibit significant enzymatic activity was a polypeptide composed ofresidues 75-247 of StxA (A1-Jaufy A et al., Infect Immun 62; 956-60(1994)).

Although Shiga toxin effector polypeptides of the present invention maycommonly be smaller than the full-length Shiga toxin A Subunit, theShiga toxin effector polypeptide of a cell-targeting molecule of thepresent invention may need to maintain the polypeptide region from aminoacid position 77 to 239 (SLT-1A (SEQ ID NO: 1) or StxA (SEQ ID NO:2)) orthe equivalent in other A Subunits of members of the Shiga toxin family(e.g. 77 to 238 of (SEQ ID NOs: 3 and 7-18)). For example, in certainembodiments of the molecules of the present invention, the Shiga toxineffector polypeptides of the present invention derived from a Shigatoxin may comprise or consist essentially of the polypeptide representedby the amino acid sequence selected from amino acids 75 to 251, 1 to241, 1 to 251, and I to 261 of any one of SEQ ID NOs: 1-2 and 4-6.Similarly, Shiga toxin effector polypeptides derived from a Shiga toxin2 may comprise or consist essentially of the polypeptide represented bythe amino acid sequence selected from amino acids 75 to 250, 1 to 241, 1to 250, and 1 to 260 of any one of SEQ ID NOs: 3 and 7-18.

Although derived from a wild-type Shiga toxin A Subunit polypeptide, forcertain embodiments of the molecules of the present invention, the Shigatoxin effector polypeptide differs from a naturally occurring Shigatoxin A Subunit by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,35, 40 or more amino acid residues (but by no more than that whichretains at least 85%, 90%, 95%, 99%, or more amino acid sequenceidentity).

The invention further provides variants of the cell-targeting moleculesof the present invention, wherein the Shiga toxin effector polypeptidediffers from a naturally occurring Shiga toxin A Subunit by only or upto 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40 or more aminoacid residues (but by no more than that which retains at least 85%, 90%o, 95%, 99% or more amino acid sequence identity). Thus, a molecule ofthe present invention derived from an A Subunit of a member of the Shigatoxin family may comprise additions, deletions, truncations, or otheralterations from the original sequence as long as at least 85%, 90%,95%, 99% or more amino acid sequence identity is maintained to anaturally occurring Shiga toxin A Subunit, such as, e.g., wherein thereis a disrupted, furin-cleavage motif at the carboxy terminus of a Shigatoxin A1 fragment derived region.

Accordingly, in certain embodiments, the Shiga toxin effectorpolypeptide of a molecule of the present invention comprises or consistsessentially of amino acid sequences having at least 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5% or 99.7% overall sequenceidentity to a naturally occurring Shiga toxin A Subunit (e.g., any oneof SEQ ID NOs: 1-18), such as, e.g., wherein there is a disrupted,furin-cleavage motif at the carboxy terminus of a Shiga toxin A1fragment derived region.

Optionally, either a full-length or a truncated version of the Shigatoxin effector polypeptide of a cell-targeting molecule of the presentof invention, wherein the Shiga toxin derived polypeptide comprises oneor more mutations (e.g. substitutions, deletions, insertions, orinversions) as compared to a naturally occurring Shiga toxin A Subunit.It is preferred in certain embodiments of the invention that the Shigatoxin effector polypeptides have sufficient sequence identity to awild-type Shiga toxin A Subunit to retain cytotoxicity after entry intoa cell, either by well-known methods of host cell transformation,transfection, infection or induction, or by internalization mediated bya cell-targeting binding region linked with the Shiga toxin effectorpolypeptide. The most critical region of a Shiga toxin A Subunit forenzymatic activity is the active site, which is positioned around aminoacid residues 137/138 to 209/210, depending on the variant, such as anyone of SEQ ID NOs: 1-18. The most critical residues for enzymaticactivity and/or cytotoxicity in the Shiga toxin A Subunits have beenmapped to the following residue-positions: asparagine-75, tyrosine-77,glutamate-167, arginine-170, and arginine-176 among others (Di R et al.,Toxicon 57: 525-39 (2011)). In any one of the embodiments of theinvention, the Shiga toxin effector polypeptides may preferably but notnecessarily maintain one or more conserved amino acids at positions,such as those found at positions 77, 167, 170, and 176 in StxA, SLT-1A,or the equivalent conserved position in other members of the Shiga toxinfamily which are typically required for potent cytotoxic activity. Thecapacity of a cytotoxic cell-targeting molecule of the present inventionto cause cell death, e.g. its cytotoxicity, may be measured using anyone or more of a number of assays well known in the art.

It should be noted that cell-targeting molecules of the invention thatcomprise Shiga toxin effector polypeptides with even considerablereductions in the Shiga toxin effector function(s) of subcellularrouting as compared to wild-type Shiga toxin effector polypeptides maystill be capable of delivering their heterologous, CD8+ T-cellepitope-peptide cargos to the MHC class I presentation pathway of atarget cell, such as, e.g., in sufficient quantities to induce an immuneresponse involving intercellular engagement of a CD8+ immune cell and/orto detect certain subcellular compartments of specific cell-types aseven presentation of a single pMHC I complex is sufficient forintercellular engagement of a presenting cell by a CTL for cytolysis(Sykulev Y et al., Immunity 4: 565-71 (1996)).

In certain embodiments of the cell-targeting molecule of the presentinvention, the Shiga toxin effector polypeptide comprises (1) a Shigatoxin A1 fragment derived polypeptide having a carboxy-terminus and (2)a disrupted furin-cleavage motif at the carboxy-terminus of the Shigatoxin A1 fragment derived polypeptide. The carboxy-terminus of a Shigatoxin A1 fragment derived polypeptide may be identified by the skilledworker by using techniques known in the art, such as, e.g., by usingprotein sequence alignment software to identify (i) a furin-cleavagemotif conserved with a naturally occurring Shiga toxin. (ii) a surfaceexposed, extended loop conserved with a naturally occurring Shiga toxin,and/or (iii) a stretch of amino acid residues which are predominantlyhydrophobic (i.e. a hydrophobic “patch”) that may be recognized by theERAD system.

The Shiga toxin effector polypeptide of the cell-targeting molecule ofthe present invention (1) may completely lack any furin-cleavage motifat a carboxy-terminus of its Shiga toxin A1 fragment region and/or (2)comprise a disrupted furin-cleavage motif at the carboxy-terminus of itsShiga toxin A1 fragment region and/or region derived from thecarboxy-terminus of a Shiga toxin A1 fragment. A disruption of afurin-cleavage motif includes various alterations to an amino acidresidue in the furin-cleavage motif, such as, e.g., a post-translationmodification(s), an alteration of one or more atoms in an amino acidfunctional group, the addition of one or more atoms to an amino acidfunctional group, the association to a non-proteinaceous moiety(ies),and/or the linkage to an amino acid residue, peptide, polypeptide suchas resulting in a branched proteinaceous structure. For example, thelinkage of a heterologous, CD8+ T-cell epitope-peptide cargo to thecarboxy-terminus of the Shiga toxin A1 fragment region of a wild-typeShiga toxin effector polypeptide may result in reduced furin-cleavage ofthe Shiga toxin effector polypeptide as compared to a reference moleculelacking the linked epitope-peptide cargo.

Protease-cleavage resistant, Shiga toxin effector polypeptides may becreated from a Shiga toxin effector polypeptide and/or Shiga toxin ASubunit polypeptide, whether naturally occurring or not, using a methoddescribed herein, described in WO 2015/191764, and/or known to theskilled worker, wherein the resulting molecule still retains one or moreShiga toxin A Subunit functions.

For purposes of the present invention with regard to a furin-cleavagesite or furin-cleavage motif, the term “disruption” or “disrupted”refers to an alteration from the naturally occurring furin-cleavage siteand/or furin-cleavage motif, such as, e.g., a mutation, that results ina reduction in furin-cleavage proximal to the carboxy-terminus of aShiga toxin A1 fragment region, or identifiable region derived thereof,as compared to the furin-cleavage of a wild-type Shiga toxin A Subunitor a polypeptide derived from a wild-type Shiga toxin A Subunitcomprising only wild-type polypeptide sequences. An alteration to anamino acid residue in the furin-cleavage motif includes a mutation inthe furin-cleavage motif, such as, e.g., a deletion, insertion,inversion, substitution, and/or carboxy-terminal truncation of thefurin-cleavage motif, as well as a post-translation modification, suchas, e.g., as a result of glycosylation, albumination, and the like whichinvolve conjugating or linking a molecule to the functional group of anamino acid residue. Because the furin-cleavage motif is comprised ofabout twenty, amino acid residues, in theory, alterations,modifications, mutations, deletions, insertions, and/or truncationsinvolving one or more amino acid residues of any one of these twentypositions might result in a reduction of furin-cleavage sensitivity(Tian S et al., Sci Rep 2: 261 (2012)).

For purposes of the present invention, a “disrupted furin-cleavagemotif” is furin-cleavage motif comprising an alteration to one or moreamino acid residues derived from the 20 amino acid residue regionrepresenting a conserved, furin-cleavage motif found in native, Shigatoxin A Subunits at the junction between the Shiga toxin A1 fragment andA2 fragment regions and positioned such that furin cleavage of a Shigatoxin A Subunit results in the production of the A1 and A2 fragments;wherein the disrupted furin-cleavage motif exhibits reduced furincleavage in an experimentally reproducible way as compared to areference molecule comprising a wild-type, Shiga toxin A1 fragmentregion fused to a carboxy-terminal polypeptide of a size large enough tomonitor furin cleavage using the appropriate assay known to the skilledworker and/or described herein.

Examples of types of mutations which can disrupt a furin-cleavage siteand furin-cleavage motif are amino acid residue deletions, insertions,truncations, inversions, and/or substitutions, including substitutionswith non-standard amino acids and/or non-natural amino acids. Inaddition, furin-cleavage sites and furin-cleavage motifs can bedisrupted by mutations comprising the modification of an amino acid bythe addition of a covalently-linked structure which masks at least oneamino acid in the site or motif, such as, e.g., as a result ofPEGylation, the coupling of small molecule adjuvants, and/orsite-specific albumination.

If a furin-cleavage motif has been disrupted by mutation and/or thepresence of non-natural amino acid residues, certain disruptedfurin-cleavage motifs may not be easily recognizable as being related toany furin-cleavage motif; however, the carboxy-terminus of the Shigatoxin A1 fragment derived region will be recognizable and will definewhere the furin-cleavage motif would be located were it not disrupted.For example, a disrupted furin-cleavage motif may comprise less than thetwenty, amino acid residues of the furin-cleavage motif due to acarboxy-terminal truncation as compared to a Shiga toxin A Subunitand/or Shiga toxin A1 fragment.

In certain embodiments of the cell-targeting molecule of the presentinvention, the Shiga toxin effector polypeptide comprises (1) a Shigatoxin A1 fragment derived polypeptide having a carboxy-terminus and (2)a disrupted furin-cleavage motif at the carboxy-terminus of the Shigatoxin A1 fragment polypeptide region; wherein the cell-targetingmolecule is more furin-cleavage resistant as compared to a referencemolecule, such as, e.g., a related molecule comprising only a wild-typeShiga toxin polypeptide component(s) or only a Shiga toxin effectorpolypeptide component (s) having a conserved, furin-cleavage motifbetween A1 and A2 fragments. For example, a reduction in furin cleavageof one molecule compared to a reference molecule may be determined usingan in vitro, furin-cleavage assay described in WO 2015/191764, conductedusing the same conditions, and then performing a quantitation of theband density of any fragments resulting from cleavage to quantitativelymeasure in change in furin cleavage.

In general, the protease-cleavage sensitivity of a cell-targetingmolecule of the present invention is tested by comparing it to the samemolecule having its furin-cleavage resistant, Shiga toxin effectorpolypeptide component(s) replaced with a wild-type, Shiga toxin effectorpolypeptide component(s) comprising a Shiga toxin A1 fragment. Incertain embodiments, the molecules of the present invention comprising adisrupted furin-cleavage motif exhibit a reduction in in vitro furincleavage of 30% o, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98% orgreater compared to a reference molecule comprising a wild-type, Shigatoxin A1 fragment fused at its carboxy-terminus to a peptide orpolypeptide.

In certain embodiments of the cell-targeting molecules of the presentinvention, the Shiga toxin effector polypeptide comprises a disruptionin one or more amino acids derived from the conserved, highlyaccessible, protease-cleavage sensitive loop of Shiga toxin A Subunits.In certain further embodiments, the Shiga toxin effector polypeptidecomprising a disrupted furin-cleavage motif comprising a mutation in thesurface-exposed, protease sensitive loop conserved among Shiga toxin ASubunits. In certain further embodiments, the mutation reduces thesurface accessibility of certain amino acid residues within the loopsuch that furin-cleavage sensitivity is reduced.

In certain embodiments, the disrupted furin-cleavage motif of a Shigatoxin effector polypeptide of a cell-targeting molecule of the presentinvention comprises a disruption in terms of existence, position, orfunctional group of one or both of the consensus amino acid residues P1and P4, such as, e.g., the amino acid residues in positions 1 and 4 ofthe minimal furin-cleavage motif R/Y-x-x-R For example, mutating one orboth of the two arginine residues in the minimal, furin consensus siteR-x-x-R to alanine will disrupt a furin-cleavage motif by reducing orabolishing furin-cleavage at that site. For example, mutating one orboth arginine residues to histidine will cause reduction in furincleavage. Similarly, amino acid residue substitutions of one or both ofthe arginine residues in the minimal furin-cleavage motif R-x-x-R to anynon-conservative amino acid residue known to the skilled worker willreduced the furin-cleavage sensitivity of the motif. In particular,amino acid residue substitutions of arginine to any non-basic amino acidresidue which lacks a positive charge, such as, e.g., A, G, P, S, T, D,E, Q, N, C, I, L, M, V, F, W, and Y, will result in a disruptedfurin-cleavage motif.

In certain embodiments, the disrupted furin-cleavage motif of a Shigatoxin effector polypeptide of the present invention comprises adisruption in the spacing between the consensus amino acid residues P4and P1 in terms of the number of intervening amino acid residues beingother than two, and, thus, changing either P4 and/or P1 into a differentposition and eliminating the P4 and/or P1 designations. For example,deletions within the furin-cleavage motif of the minimal furin-cleavagesite or the core, furin-cleavage motif will reduce the furin-cleavagesensitivity of the furin-cleavage motif.

In certain embodiments of the cell-targeting molecules of the presentinvention, the disrupted furin-cleavage motif comprises one or moreamino acid residue substitutions, as compared to a wild-type, Shigatoxin A Subunit. In certain further embodiments, the disruptedfurin-cleavage motif comprises one or more amino acid residuesubstitutions within the minimal furin-cleavage site R/Y-x-x-R, such as,e.g., for StxA and SLT-1A derived Shiga toxin effector polypeptides, thenatively positioned amino acid residue R248 substituted with anynon-positively charged, amino acid residue and/or R251 substituted withany non-positively charged, amino acid residue; and for SLT-2A derivedShiga toxin effector polypeptides, the natively positioned amino acidresidue Y247 substituted with any non-positively charged, amino acidresidue and/or R250 substituted with any non-positively charged, aminoacid residue.

In certain embodiments of the cell-targeting molecules of the presentinvention, the disrupted furin-cleavage motif comprises an un-disrupted,minimal furin-cleavage site R/Y-x-x-R but instead comprises a disruptedflanking region, such as, e.g., amino acid residue substitutions in oneor more amino acid residues in the furin-cleavage motif flanking regionsnatively position at, e.g., 241-247 and/or 252-259. In certain furtherembodiments, the disrupted furin cleavage motif comprises a substitutionof one or more of the amino acid residues located in the P1-P6 region ofthe furin-cleavage motif; mutating P1′ to a bulky amino acid, such as,e.g., R, W, Y, F, and H; and mutating P2′ to a polar and hydrophilicamino acid residue; and substituting one or more of the amino acidresidues located in the P1′-P6′ region of the furin-cleavage motif withone or more bulky and hydrophobic amino acid residues

In certain embodiments of the cell-targeting molecules of the presentinvention, the disrupted furin-cleavage motif comprises a deletion,insertion, inversion, and/or substitution of at least one amino acidresidue within the furin-cleavage motif relative to a wild-type Shigatoxin A Subunit. In certain further embodiments, the disruptedfurin-cleavage motif comprises a disruption of the amino acid sequencenatively positioned at 248-251 of the A Subunit of Shiga toxin (SEQ IDNOs: 1-2 and 4-6), at 247-250 of the A Subunit of Shiga-like toxin 2(SEQ ID NOs: 3 and 7-18), or at the equivalent position in a conservedShiga toxin effector polypeptide and/or non-native Shiga toxin effectorpolypeptide sequence. In certain further embodiments, the disruptedfurin-cleavage motif comprises a disruption which comprises a mutation,such as, e.g., an amino acid substitution to a non-standard amino acidor an amino acid with a chemically modified side chain. In certainfurther embodiments, the disrupted furin-cleavage motif comprisescomprise a disruption which comprises a deletion of at least one aminoacid within the furin-cleavage motif. In certain further embodiments,the disrupted furin-cleavage motif comprises the deletion of nine, ten,eleven, or more of the carboxy-terminal amino acid residues within thefurin-cleavage motif. In these embodiments, the disrupted furin-cleavagemotif will not comprise a furin-cleavage site or a minimalfurin-cleavage motif. In other words, certain embodiments lack afurin-cleavage site at the carboxy-terminus of the A1 fragment region.

In certain embodiments of the cell-targeting molecules of the presentinvention, the disrupted furin-cleavage motif comprises an amino acidresidue deletion and an amino acid residue substitution as well as acarboxy-terminal truncation as compared to a wild-type, Shiga toxin ASubunit. In certain further embodiments, the disrupted furin-cleavagemotif comprises one or more amino acid residue deletions andsubstitutions within the minimal furin-cleavage site R/Y-x-x-R.

In certain embodiments of the cell-targeting molecules of the presentinvention, the disrupted furin-cleavage motif comprises both an aminoacid substitution within the minimal furin-cleavage site R/Y-x-x-R and acarboxy-terminal truncation as compared to a wild-type, Shiga toxin ASubunit, such as, e.g., for StxA and SLT-1A derived Shiga toxin effectorpolypeptides, truncations ending at the natively amino acid position249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276,277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,291, or greater and comprising the natively positioned amino acidresidue R248 and/or R251 substituted with any non-positively charged,amino acid residue where appropriate; and for SLT-2A derived Shiga toxineffector polypeptides, truncations ending at the natively amino acidposition 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259,260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273,274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287,288, 289, 290, 291, or greater and comprising the natively positionedamino acid residue Y247 and/or R250 substituted with any non-positivelycharged, amino acid residue where appropriate.

In certain embodiments of the cell-targeting molecules of the presentinvention, the disrupted furin-cleavage motif comprises both an aminoacid residue deletion and an amino acid residue substitution as comparedto a wild-type, Shiga toxin A Subunit. In certain further embodiments,the disrupted furin-cleavage motif comprises one or more amino acidresidue deletions and substitutions within the minimal furin-cleavagesite R/Y-x-x-R.

In certain embodiments of the cell-targeting molecule of the presentinvention, the disrupted furin-cleavage motif comprises an amino acidresidue deletion, an amino acid residue insertion, an amino acid residuesubstitution and/or a carboxy-terminal truncation as compared to awild-type, Shiga toxin A Subunit.

The cell-targeting molecules of the present invention each comprise oneor more, heterologous, CD8+ T-cell epitope-peptide cargos which is notembedded or inserted in the Shiga toxin A1 fragment region of any Shigatoxin effector polypeptide component. In certain embodiments, the CD8+T-cell epitope-peptide is an antigenic and/or immunogenic epitope in ahuman. In certain embodiments, the CD8+ T-cell epitope-peptide componentof the cell-targeting molecules of the present invention comprises orconsists essentially of an 8-11 amino acid long peptide derived from amolecule of a microbial pathogen which infects humans, such as, e.g., anantigen from a virus that infects humans. In certain furtherembodiments, the CD8+ T-cell epitope-peptide component of thecell-targeting molecules of the invention comprises or consistsessentially of any one of the peptides shown in SEQ ID NOs: 19-28.

In certain embodiments of the cell-targeting molecules of the presentinvention, the heterologous, CD8+ T-cell epitope-peptide cargo is linkedto the cell-targeting molecule via a thiol linkage, such as, e.g.,disulfide bond. In certain further embodiments, the thiol linkage is acysteine to cysteine disulfide bond.

In certain embodiments of the cell-targeting molecules of the presentinvention, the heterologous, CD8+ T-cell epitope-peptide cargo is linkedto the cell-targeting molecule via a disulfide bond involving thefunctional group of a cysteine residue of a Shiga toxin effectorpolypeptide component of the cell-targeting molecule, such as, e.g.,C241 of SLT-2A (SEQ ID NO:3) or 242 of StxA (SEQ ID NO:2) or SLT-1A (SEQID NO: 1). In certain further embodiments, the cysteine residue ispositioned carboxy-terminal to the carboxy terminus of the Shiga toxinA1 fragment region of the Shiga toxin effector polypeptide (e.g., thecysteine residue C260 of SLT-2A (SEQ ID NO:3) or C261 of StxA (SEQ IDNO:2) or SLT-1A (SEQ ID NO: 1)).

The cell-targeting molecules of the present invention comprise at leastone cell-targeting binding region. Among certain embodiments of thecell-targeting molecules of the present invention, the binding region isderived from an immunoglobulin-type polypeptide selected for specificand high-affinity binding to a surface antigen on the cell surface of acancer or tumor cell, where the antigen is restricted in expression tocancer or tumor cells (see Glokler J et al., Molecules 15: 2478-90(2010); Liu Y et al., Lab Chip 9: 1033-6 (2009). In accordance withother embodiments, the binding region is selected for specific andhigh-affinity binding to a surface antigen on the cell surface of acancer cell, where the antigen is over-expressed or preferentiallyexpressed by cancer cells as compared to non-cancer cells. Somerepresentative target biomolecules include, but are not limited to, thefollowing enumerated targets associated with cancers and/or specificimmune cell-types.

Many immunoglobulin-type binding regions that bind with high affinity toextracellular epitopes associated with cancer cells are known to theskilled worker, such as binding regions that bind any one of thefollowing target biomolecules: annexin AI, B3 melanoma antigen, B4melanoma antigen, CD2, CD3, CD4, CD19, CD20 (B-lymphocyte antigenprotein CD20), CD22, CD25 (interleukin-2 receptor IL2R), CD30 (TNFRSF8),CD37, CD38 (cyclic ADP ribose hydrolase), CD40, CD44 (hyaluronanreceptor), ITGAV (CD51), CD56, CD66, CD70, CD71 (transferrin receptor),CD73, CD74 (HLA-DR antigens-associated invariant chain), CD79, CD98,endoglin (END, CD105), CD106 (VCAM-1), CD138, chemokine receptor type 4(CDCR-4, fusin, CD184), CD200, insulin-like growth factor I receptor(CD221), mucinl (MUC1, CD227, CA6, CanAg), basal cell adhesion molecule(B-CAM, CD239), CD248 (endosialin, TEMI), tumor necrosis factor receptor10b (TNFRSF10B, CD262), tumor necrosis factor receptor 13B (TNFRSFI3B,TACI, CD276), vascular endothelial growth factor receptor 2 (KDR,CD309), epithelial cell adhesion molecule (EpCAM, CD326), humanepidermal growth factor receptor 2 (HER2, Neu, ErbB2, CD340), cancerantigen 15-3 (CA15-3), cancer antigen 19-9 (CA 19-9), cancer antigen 125(CA125, MUC16), CA242, carcinoembryonic antigen-related cell adhesionmolecules (e.g. CEACAM3 (CD66d) and CEACAM5), carcinoembryonic antigenprotein (CEA), choline transporter-like protein 4 (SLC44A4), chondroitinsulfate proteoglycan 4 (CSP4, MCSP, NG2). CTLA4, delta-like proteins(e.g. DLL3, DLL4), ectonucleotide pyrophosphatase/phosphodiesteraseproteins (e.g. ENPP3), endothelin receptors (ETBRs), epidermal growthfactor receptor (EGFR, ErbB1), folate receptors (FOLRs, e.g. FRa), G-28,ganglioside GD2, ganglioside GD3, HLA-Dr100, HLA-DRB, human epidermalgrowth factor receptor 1 (HERI), HER3/ErbB-3, Ephrin type-B receptor 2(EphB2), epithelial cell adhesion molecule (EpCAM), fibroblastactivation protein (FAP/seprase), guanylyl cyclase c (GCC), insulin-likegrowth factor 1 receptor (IGF1R), interleukin 2 receptor (IL-2R),interleukin 6 receptor (IL-6R), integrins alpha-V beta-3 (av3),integrins alpha-V beta-5 (av05), integrins alpha-5 beta-1 (as i), L6,zinc transporter (LIV-1), MPG, melanoma-associated antigen I protein(MAGE-1), melanoma-associated antigen 3 (MAGE-3), mesothelin (MSLN),metalloreductase STEAPI, MPG, MS4A, NaPi2b, nectins (e.g. nectin-4),p21, p97, polio virus receptor-like 4 (PVRL4),protease-activated-receptors (such as PARI), prostate-specific membraneantigen proteins (PSMAs), SLIT and NTRK-like proteins (e.g. SLITRK6),Thomas-Friedenreich antigen, transmembrane glycoprotein (GPNMB),trophoblast glycoproteins (TPGB, 5T4. WAIFI), and tumor-associatedcalcium signal transducers (TACSTDs, e.g. Trop-2, EGP-1, etc.) (see e.g.Lui B et al., Cancer Res 64: 704-10 (2004); Novellino L et al., CancerImmunol Immunother 54: 187-207 (2005): Bagley R et al., Int J Oncol 34:619-27 (2009); Gerber H et al., mAbs 1: 247-53 (2009): Beck A et al.,Nat Rev Ihnmunol 10: 345-52 (2010); Andersen J et al., J Biol Chem 287:22927-37 (2012); Nolan-Stevaux O et al., PLoS One 7: e50920 (2012): RustS et al., Mol Cancer 12: 11 (2013)). This list of target biomolecules isintended to be non-limiting. It will be appreciated by the skilledworker that any desired target biomolecule associated with a cancer cellor other desired cell-type may be used to design or select a bindingregion which may be suitable for use as a component of a cell-targetingmolecule of the present invention.

Examples of other target biomolecules which are strongly associated withcancer cells and are bound with high-affinity by a knownimmunoglobulin-type binding region include BAGE proteins (B melanomaantigens), basal cell adhesion molecules (BCAMs or Lutheran blood groupglycoproteins), bladder tumor antigen (BTA), SAIL (C16orf54),cancer-testis antigen NY-ESO-1, cancer-testis antigen LAGE proteins,CD19 (B-lymphocyte antigen protein CD19), CD21 (complement receptor-2 orcomplement 3d receptor), CD26 (dipeptidyl peptidase-4, DPP4, oradenosine deaminase complexing protein 2), CD33 (sialic acid-bindingimmunoglobulin-type lectin-3), CD52 (CAMPATH-1 antigen), CD56, CS1 (SLAMfamily number 7 or SLAMF7), cell surface A33 antigen protein (gpA33),Epstein-Barr virus antigen proteins, GAGE/PAGE proteins (melanomaassociated cancer/testis antigens), hepatocyte growth factor receptor(HGFR or c-Met), MAGE proteins, melanoma antigen recognized by T-cells 1protein (MART-1/MelanA. MARTI), mucins, Preferentially Expressed Antigenof Melanoma (PRAME) proteins, prostate specific antigen protein (PSA),prostate stem cell antigen protein (PSCA), Receptor for AdvancedGlycation Endproducts (RAGE), tumor-associated glycoprotein 72 (TAG-72),vascular endothelial growth factor receptors (VEGFRs), and Wilms' tumorantigen.

Examples of other target biomolecules which are strongly associated withcancer cells are carbonic anhydrase IX (CA9/CAIX), claudin proteins(CLDN3, CLDN4), ephrin type-A receptor 3 (EphA3), folate bindingproteins (FBP), ganglioside GM2, insulin-like growth factor receptors,integrins (such as CD11a-c), receptor activator of nuclear factor kappaB (RANK), receptor tyrosine-protein kinase erB-3, tumor necrosis factorreceptor 10A (TRAIL-R1/DR4), tumor necrosis factor receptor 10B(TRAIL-R2), tenascin C, and CD64 (FcγRI) (see Hough C et al., Cancer Res60: 6281-7 (2000); Thepen T et al., Nat Biotechnol 18: 48-51 (2000);Pastan I et al., Nat Rev Cancer 6: 559-65 (2006); Pastan, Annu Rev Med58: 221-37 (2007); Fitzgerald D et al., Cancer Res 71: 6300-9 (2011);Scott A et al., Cancer Immun 12: 14-22 (2012)). This list of targetbiomolecules is intended to be non-limiting.

In addition, there are numerous other examples of contemplated, targetbiomolecules, such as, e.g., ADAM metalloproteinases (e.g ADAM-9,ADAM-10, ADAM-12, ADAM-15, ADAM-17), ADP-ribosyltransferases (ART1,ART4), antigen F4/80, bone marrow stroma antigens (BST1, BST2), breakpoint cluster region-c-abl oncogene (BCR-ABL) proteins, C3aR (complementcomponent 3a receptors), CD7, CD13, CD14, CDl5 (Lewis X orstage-specific embryonic antigen 1), CD23 (FC epsilon RII), CD45(protein tyrosine phosphatase receptor type C), CD49d, CD53, CD54(intercellular adhesion molecule 1), CD63 (tetraspanin), CD69, CD80,CD86, CD88 (complement component 5a receptor 1), CD115 (colonystimulating factor 1 receptor), IL-R (interleukin-1 receptor), CD123(interleukin-3 receptor), CD129 (interleukin 9 receptor), CD183(chemokine receptor CXCR3), CD191 (CCR1), CD193 (CCR3), CD195 (chemokinereceptor CCR5), CD203c, CD225 (interferon-induced transmembrane protein1), CD244 (Natural Killer Cell Receptor 2B4), CD282 (Toll-like receptor2), CD284 (Toll-like receptor 4), CD294 (GPR44), CD305(leukocyte-associated immunoglobulin-like receptor 1), ephrin type-Areceptor 2 (EphA2), FceRIa, galectin-9, alpha-fetoprotein antigen 17-A1protein, human aspartyl (asparaginyl) beta-hydroxylase (HAAH),immunoglobulin-like transcript ILT-3, lysophosphatidlglycerolacyltransferase 1 (LPGAT1/IAA0205), lysosome-associated membraneproteins (LAMPs, such as CD107), melanocyte protein PMEL (gp100),myeloid-related protein-14 (mrp-14), NKG2D ligands (e.g., MICA, MICB,ULBP1, ULBP2, UL-16-binding proteins. H-60s, Rae-1s, and homologsthereof), receptor tyrosine-protein kinase erbB-3, SART proteins,scavenger receptors (such as CD64 and CD68), Siglecs (sialicacid-binding immunoglobulin-type lectins), syndecans (such as SDC I orCD138), tyrosinase, tyrosinease-related protein 1 (TRP-1),tyrosinease-related protein 2 (TRP-2), tyrosinase associated antigen(TAA), APO-3, BCMA, CD2, CD3, CD4, CD8, CD18, CD27, CD28, CD29, CD41,CD49, CD90, CD95 (Fas), CD103, CD104, CD134 (OX40), CD137 (4-1BB), CD152(CTLA-4), chemokine receptors, complement proteins, cytokine receptors,histocompatibility proteins, ICOS, interferon-alpha, interferon-beta,c-myc, osteoprotegerin, PD-I, RANK, TACI, TNF receptor superfamilymember (TNF-R1, TNFR-2), Apo2/TRAIL-R1, TRAIL-R2, TRAIL-R3, and TRAIL-R4(see Scott A et al., Cancer Immunity 12: 14 (2012): Cheever M et al.,Clin Cancer Res 15: 5323-37 (2009)), for target biomolecules and notethe target biomolecules described therein are non-limiting examples).

In certain embodiments, the binding region comprises or consistsessentially of an immunoglobulin-type binding region capable ofspecifically binding with high-affinity to the cellular surface of acell-type of the immune system. For example, immunoglobulin-type bindingdomains are known which bind to immune cell surface factors, such as,e.g., CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11, CD12,CD13, CD14, CD15, CD16, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24,CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD33, CD34, CD35, CD36, CD37,CD38, CD40, CD41, CD56, CD61, CD62. CD66, CD95, CD117, CD123, CD235,CD146, CD326, interleukin-1 receptor (IL-1R), interleukin-2 receptor(IL-2R), receptor activator of nuclear factor kappa B (RANKL),SLAM-associated protein (SAP), and TNFSF18 (tumor necrosis factor ligand18 or GITRL).

For further examples of target biomolecules and binding regionsenvisioned for use in the molecules of the present invention, see WO2005/92917, WO 2007/033497, US2009/156417, JP4339511, EP 1727827,DE602004027168, EP1945660, JP4934761, EP2228383, US2013/196928, WO2014/164680, WO 2014/164693, WO 2015/138435, WO 2015/138452, WO2015/113005, WO 2015/113007, WO 2015/191764, US2015/259428, 62/168,758,62/168,759, 62/168,760, 62/168,761, 62/168,762, 62/168,763, andPCT/US2016/016580.

Certain embodiments of the cell-targeting molecules of the presentinvention are cytotoxic, cell-targeting, fusion proteins. Certainfurther embodiments are the cell-targeting molecules which comprise orconsist essentially of one of the polypeptides shown in SEQ ID NOs:19-255 and 288-748. Certain further embodiments are the cell-targetingmolecules which comprise or consist essentially of one of thepolypeptides shown in SEQ ID NOs: 252-255 and 288-748.

In certain embodiments, the cell-targeting molecule of the presentinvention is a fusion protein, such as, e.g. immunotoxins orligand-toxin fusion. Certain embodiments of the cell-targeting moleculesof the present invention are reduced-cytotoxicity or non-cytotoxic,cell-targeting, fusion proteins. Certain embodiments are thecell-targeting molecules which comprise or consist essentially of one ofthe polypeptides shown in SEQ ID NOs: 252-255 and 288-748 which furthercomprises one or more amino acid substitutions in the Shiga toxineffector polypeptide component(s) altering the natively positionedresidue selected from the group consisting of: A231E, N75A, Y77S, Y114S,E167D, R170A, R176K and/or W203A in SEQ ID NO: 1, SEQ ID NO:2, or SEQ IDNO:3, or the equivalent amino acid residue in a Shiga toxin A Subunit.Certain further embodiments are the cell-targeting molecules whichcomprise or consist essentially of one of the polypeptides shown in SEQID NOs: 258, 260, 268, 270, and 278.

To alter the half-life of a cell-targeting molecule of the presentinvention after administration to a chordate, a polyethylene glycolmolecule(s), an immunoglobulin Fc region(s), an immunoglobulin constantdomain, an inactivated Fc region(s), and/or a salvage receptor bindingepitope(s) may be incorporated into the molecule using standardtechniques known to the skilled worker (see e.g. U.S. Pat. Nos.5,739,277; 7,083,784; 7,348,004; US2008/422112; WO2010/033279:WO2013/012733; U.S. Pat. No. 9,790,268).

In certain embodiments of the cell-targeting molecule of the presentinvention, two or more binding regions, each capable of binding anextracellular part of the same target biomolecule, are associated with aShiga toxin effector polypeptide(s) to produce multivalentcell-targeting molecules of the present invention.

In certain embodiments, the multivalent cell-targeting molecules of thepresent invention comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, or 22 individual binding regions.

The multivalent cell-targeting molecules of the invention may comprisevarious multivalent structures. For example, multivalent structures canbe created using covalent and/or non-covalent interactions to associatetogether various components to form a multivalent cell-targetingmolecule of the present invention.

In certain embodiments, the multivalent cell-targeting molecules of thepresent invention are multimeric complexes of two or more proteinaceoussubunits, such as, e.g. dimers, trimers, tetramers, diabodies,triabodies, tetrabodies, etc.

In certain embodiments, the multivalent cell-targeting molecule of thepresent invention comprises two or more binding regions because two ormore components of the multivalent cell-targeting molecule arechemically linked or conjugated together. For example, chemical linkersmay be used to conjugate two or more target-binding proteins together toform a multivalent cell-targeting molecule (see e.g. Wolff E et al.,Cancer Res 53: 2560-5 (1993); Ghetie M et al., Proc Natl Acad Sci USA94: 7509-14 (1997); Ghetie M et al., Blood 97: 1392-8 (2001)).

Certain embodiments of the multivalent cell-targeting molecules of thepresent invention comprise a multimeric structure comprising two or morecomponent molecules, which may be identical or non-identical. As usedherein, the nomenclature (X)_(n) refers to a molecule comprising orconsisting of integer number (n) copies of a component (X). For example,a dimeric protein comprising two identical, monovalent, target-bindingpolypeptide subunits may be referred to as a homodimer or(target-binding monomer)₂. Another example is a mixture of multivalentproteins, each protein comprising three or more identical polypeptide“X” subunits, which is referred to herein as (X)_(2+n), where “n” refersto a positive integer and the value of “2+n” representing the number ofbinding regions per protein of the protein molecules present, thusdescribing a plurality of different multivalent protein species presentin a single protein composition.

Certain embodiments of the multivalent cell-targeting molecules of thepresent invention are multimeric, being comprised of two or moretarget-binding molecules, such as, e.g., homodimers, homotrimers, andhomotetramers, and the like. For example, two or more monovalenttarget-binding polypeptides may be combined to form multivalentcell-targeting molecules of the present invention.

In certain embodiments, the multivalent cell-targeting molecule of thepresent invention comprises two or more components, each comprising atleast one of the binding regions of the multivalent cell-targetingmolecule, because of a non-covalent intermolecular association(s)resulting from domain swapping between the two or more components whichresults in a multivalent cell-targeting molecule with a multimericstructure (see e.g. FIG. 1B). For example, protein domain swappingbetween immunoglobulin domains can be engineered and optimized as amechanism of producing precise multimeric structures (see e.g. HolligerP et al., Proc Natl Acad Sci USA 90: 6444-8 (1993); Amdt K et al.,Biochemistry 37: 12918-26 (1998)).

The skilled worker can engineer multimeric, multivalent cell-targetingmolecules of the present invention using various scFv-based polypeptideinteractions, such as, e.g. scFv-based dimeric, trimeric, tetramericcomplexes, etc. For example, the length of the linker in the scFv canaffect the spontaneous assembly of non-covalent based, multimeric,multivalent structures. Generally, linkers of twelve amino acids orless, including the absence of any linker, promote the multimerizationof polypeptides or proteins comprising scFvs into higher molecularweight species via favoring intermolecular domain swapping overintra-chain domain pairing (see e.g., Huston J et al., Methods Enzymol203: 46-88 (1991); Holliger P et al., Proc Natl Acad Sci USA 90: 6444-8(1993): Stemmer W et al., Biotechniques 14: 256-65 (1993); Whitlow M etal., Protein Eng 6: 989-95 (1993): Desplancq D et al., Protein Eng 7:1027-33 (1994): Whitlow M et al., Protein Eng 7: 1017-26 (1994); AlfthanK et al., Protein Eng 8: 725-31 (1995); Iliades P et al., FEBS Lett 409:437-41 (1997); Kortt A et al., Biomol Eng 18: 95-108 (2001); TodorovskaA et al., J Immunol Methods 248: 47-66 (2001); Tomlinson I, Holliger Pet al., Methods Enzmol 326: 461-79 (2001); Dolezal O et al., Protein Eng16: 47-56 (2003)). However, scFvs with no linker at all or a linker withan exemplary length of 15 amino acid residues may multimerize (Whitlow Met al., Protein Eng 6: 989-95 (1993): Desplancq D et al., Protein Eng 7:1027-33 (1994); Whitlow M et al., Protein Eng 7, 1017-26 (1994); AlfthanK et al., Protein Eng 8: 725-31 (1995)). The skilled worker can identifythe multimeric structure(s) created and/or purified using techniquesknown in the art and/or described herein.

In addition, engineered structures with additional covalent bonds can beused to stabilize multimeric structures that spontaneously assemble (seee.g. Glockshuber R et al., Biochemistry 29: 1362-7 (1990)). For example,the introduction of cysteine residues at specific locations may be usedto create disulfide-stabilized structures like Cys-diabodies, scFv′multimers, V_(H)H multimers, V_(NAR) multimers, and IgNAR multimers suchas, e.g., by adding the following amino acid residues: GGGGC and SGGGGC(Tai M et al., Biochemistry 29: 8024-30 (1990): Caron P et al., J ExpMed 176: 1191-5 (1992); Shopes B, J Immunol 148: 2918-22 (1992): Adams Get al., Cancer Res 53: 4026-34 (1993); McCartney J et al., Protein Eng18: 301-14 (1994): Perisic O et al., Structure 2: 1217-26 (1994): GeorgeA et al., Proc Natl Acad Sci USA 92: 8358-62 (1995); Tai M et al.,Cancer Res (Suppl) 55: 5983-9 (1995): Olafsen T et al., Protein Eng DesSel 17: 21-7 (2004)). Thus, the skilled worker can create or stabilizemultivalent cell-targeting molecules of the present invention usingdisulfide bridge(s) and/or by adding or removing cysteine residue(s) atcertain positions to control the position(s) of certain disulfidebridges.

In certain embodiments, the multivalent structure of a target-bindingmolecule of the present invention comprises two or more immunoglobulindomains that binding an extracellular part of the same targetbiomolecule. In certain embodiments, the multivalent cell-targetingmolecule of the present invention may comprise or consist of a single,continuous, polypeptide chain. For example, single-chain bivalent scFvs,sometimes referred to as tandem scFvs (taFvs), single chain diabodies(scDbs), and tandem diabodies (tanDbs or Tandabs), represent multivalentbinding proteins which are created from a single continuous polypeptide(see e.g. Mack M et al., Proc Natl Acad Sci USA 92: 7021-5 (1995);Kipriyanov S et al., J Mol Biol 293: 41-56 (1999); Cochlovius, B et al.,Cancer Res 60: 4336-41 (2000); Völkel T et al., Protein Eng 14: 815-23(2001): Jendreyko N et al., J Biol Chem 278: 47812-9 (2003): KipriyanovS et al., J Mol Biol 330: 99-111 (2003); Miller K et al., J Immunol 170:4854-61 (2003): Meng R et al., Clin Cancer Res 10: 1274-81 (2004);Schlereth B et al., Cancer Res 65: 2882-9 (2005); Huang T. Morrison S, JPharmacol Exp Ther 316: 983-91 (2006): Liu X et al., Int Immunopharmacol6: 791-9 (2006): Shen J et al., J Biol Chem 281: 10706-14 (2006); Shen Jet al., J Immunol Methods 318: 65-74 (2007); Wu C et al., Nat Biotech25: 1290-7 (2007); Li B et al., Cancer Res 68: 2400-8 (2008)).

In certain embodiments, the multivalent cell-targeting molecule of thepresent invention comprises both a linker(s) between two or more bindingregions as well as one or more disulfide bonds between components of thebinding regions, whether proximal or distal to the linker, such as adisulfide bond between two immunoglobulin regions which requires animmunoglobulin domain swapping association between those twoimmunoglobulin regions (see e.g. Glockshuber R et al., Biochemistry. 29:1362-7 (1990)).

Alternatively, two or more polypeptide chains may be linked togetherusing peptide and/or polypeptide domains which self-associate ormultimerize with each other (see e.g. U.S. Pat. No. 6,329,507; Wang L etal., Protein Eng Des Sel 26: 417-23 (2013)). For example, the additionof carboxy-terminal multimerization domains has been used to constructmultivalent proteins comprising immunoglobulin domains, such as, e.g.,scFvs, autonomous V_(H) domains, V_(H)Hs, V_(NAR)s, and IgNARs. Examplesof self-associating domains known to the skilled worker includeimmunoglobulin constant domains (such as knobs-into-holes, electrostaticsteering, and IgG/IgA strand exchange), immunoglobulin Fab chains (e.g.(Fab-scFv)2 and (Fab′ scFv)₂), immunoglobulin Fc domains (e.g.(scDiabody-Fc)2, (scFv-Fc)2 and scFv-Fc-scFv), immunoglobulin CHXdomains, immunoglobulin CH1-3 regions, immunoglobulin CH3 domains (e.g.(scDiabody-CH3)₂, LD minibody, and Flex-minibody), immunoglobulin CH4domains, CHCL domains, amphiphilic helix bundles (e.g. scFv-HLX),helix-turn-helix domains (e.g. scFv-dHlx), coiled-coil structuresincluding leucine zippers and cartilage oligometric matrix proteins(e.g. scZIP), cAMP-dependent protein kinase (PKA) dimerization anddocking domains (DDDs) combined with an A kinase anchor protein (AKAP)anchoring domain (AD) (also referred to as “dock-and-lock” or “DNL”),streptavidin, verotoxin B multimerization domains, tetramerizationregions from p53, and barnase-barstar interaction domains (Pack P,Plückthun A, Biochemistry 31: 1579-84 (1992): Holliger P et al., ProcNatl Acad Sci USA 90: 6444-8 (1993); Kipriyanov S et al., Hum AntibodiesHybridomas 6: 93-101 (1995); de Kruif J, Logtenberg T, J Biol Chem 271:7630-4 (1996); Hu S et al., Cancer Res 56: 3055-61 (1996): Kipriyanov Set al., Protein Eng 9: 203-11 (1996); Rheinnecker M et al., J Immunol157: 2989-97 (1996); Tershkikh A et al., Proc Natl Acad Sci USA 94:1663-8 (1997); Müller K et al., FEBS Lett 422: 259-64 (1998): Cloutier Set al., Mol Immunol 37: 1067-77 (2000); Li S et al., Cancer ImmunolImmunother 49: 243-52 (2000); Schmiedl A et al., Protein Eng 13: 725-34(2000); Schoonjans R et al., J Immunol 165: 7050-7 (2000): Borsi L etal., Int J Cancer 102: 75-85 (2002); Deyev S et al., Nat Biotechnol 21:1486-92 (2003); Wong W, Scott J, Nat Rev Mol Cell Biol 5: 959-70 (2004);Zhang J et al., J Mol Biol 335: 49-56 (2004); Baillie G et al., FEBSLetters 579: 3264-70 (2005); Rossi E et al., Proc Natl Acad Sci USA 103:6841-6 (2006); Simmons D et al., J Immunol Methods 315: 171-84 (2006):Braren I et al., Biotechnol Appl Biochem 47: 205-14 (2007); Chang C etal., Clin Cancer Res 13: 5586-91 s (2007); Liu M et al., Biochem J 406:237-46 (2007); Zhang J et al., Protein Expr Purif 65; 77-82 (2009); BellA et al., Cancer Lett 289: 81-90 (2010); Iqbal U et al., Br J Pharmacol160: 1016-28 (2010); Asano R et al., FEBS J 280: 4816-26 (2013): Gil D.Schrum A, Adv Biosci Biotechnol 4: 73-84 (2013)).

In certain embodiments, the structure of a multivalent cell-targetingmolecule of the present invention is engineered from an antibody or Fabfragment. For example, multivalent cell-targeting molecules may beengineered using approaches known to the skilled worker (see e.g.Shuford W et al., Science 252: 724-7 (1991); Caron P et al., J Exp Med176: 1191-5 (1992): Shopes B, J Immunol 148: 2918-22 (1992); Wolff E etal., Cancer Res 53: 2560-5 (1993)).

In certain embodiments of the multivalent cell-targeting molecules ofthe present invention, all the cell-targeting binding regions of themultivalent cell-targeting molecules are identical and/or share the samebinding specificities. In such embodiments, the multivalentcell-targeting molecule of the invention is monospecific-meaning itcomprises binding regions that bind with high affinity to the sameextracellular target biomolecule, overlapping extracellular epitopes inthe same target biomolecule, and/or the same extracellular epitope in atarget biomolecule. Whether two binding regions are binding to the sameextracellular part of a target biomolecule may be determined by theskilled worker with available methods, such as, e.g., empirically usingcompetitive binding assays or predictively based on the overlap of knownepitope and/or immunized peptide sequences.

In certain embodiments, the multivalent cell-targeting molecule of thepresent invention may comprise binding regions that bind with highaffinity to non-identical epitopes, whether non-overlapping oroverlapping. The multivalent cell-targeting molecules of the presentinvention may comprise binding regions with high binding affinity tonon-overlapping epitopes. Multispecific, multivalent cell-targetingmolecules of the present invention may be created using two or moredifferent binding regions, such as, e.g., two different scFvs, V_(H)Hs,V_(NAR)s, and/or IgNARs in diabodies, triabodies, tandem formats(including tandem di-scFv, tandem tri-scFv, and scFv-Fc tandems),single-chain diabodies (scDb), tandem Fvs, bispecific scFv (Bis-scFv),scFv2, (Fab′)₃, tetrameric (scFv2)₂, scFv2-Fc, and combinations ofscFvs, V_(H)HS, V_(NAR)s, and/or IgNARs with different specificities(Adams G et al., Cancer Res 53: 4026-34 (1993): Mallender W et al., JBiol Chem 269: 199-206 (1994): Todorovska A et al., J Immunol Methods248: 47-66 (2001); Korn T et al., J Gene Med 6: 642-51 (2004); Lu D etal., J Biol Chem 280: 19665-72 (2005); Schneider M et al., Eur J Immunol35: 987-95 (2005): Wittel U et al., Nucl Med Biol 32: 157-64 (2005);Semenyuk E et al., Biochimie 89: 31-8 (2007)).

In certain embodiments, the multivalent cell-targeting molecule of thepresent invention may comprise a single, continuous polypeptidecomponent which is multimerized with itself or another protein to form amultimeric structure. For example, single-chain bivalent scFvs,sometimes referred to as tandem scFvs (taFvs), single chain diabodies(scDbs), and tandem diabodies (tanDbs or Tandabs), can be expressed assingle continuous polypeptide chain (Mack M et al., Proc Natl Acad SciUSA 92: 7021-5 (1995): Kipriyanov S et al., J Mol Biol 293: 41-56(1999); Cochlovius, B et al., Cancer Res 60: 4336-41 (2000); Volkel T etal., Protein Eng 14: 815-23 (2001): Kipriyanov S et al., J Mol Biol 330:99-111 (2003); Schlereth B et al., Cancer Res 65: 2882-9 (2005)). Thesemultivalent structures may be engineered to multimerize intohigher-order, higher-valence structures, such as, e.g. a tetravalentF(ab′)2, (taFv)2, and (scDb)2 structures (see Todorovska A et al., JImmunol Methods 248: 47-66 (2001)).

Structures comprising two scFvs linked by non-covalent interactions dueto the intermolecular pairing of variable regions are known to theskilled worker, such as, e.g., diabodies, mini-antibodies, and bivalentmini-antibodies, all of which may be either monospecific or bispecific(Holliger, P et al., Proc Natl Acad Sci USA 90: 6444-8 (1993): Pack P etal., Biotechnology (NY) 11: 1217-7 (1993); Tai M et al., Cancer Res(Suppl) 55: 5983-9 (1995); Atwell J et al., Mol Inmunol 33: 1301-12(1996); Rheinnecker M et al., J Immunol 157: 2989-97 (1996); Schier R etal., J Mol Biol 255: 28-43 (1996); Adams G et al., Br J Cancer 77:1405-12 (1998): Todorovska A et al., J Immunol Methods 248: 47-66(2001): Buihler P et al., Cancer Immunol Imnmunother 57: 43-52 (2008)).Numerous scFv monomers have been observed to naturally form multimers oroligomers (e.g. diabodies, triabodies, and tetrabodies) due toself-association, with the majority form being dimeric for scFvstructures comprising linkers of 3-12 amino acid residues (Essig N etal., J Mol Biol 234: 897-901 (1993): Griffiths A et al., EMBO J 12:725-34 (1993); Holliger P et al., Proc Natl Acad Sci (USA 90: 6444-8(1993); Whitlow M et al., Protein Eng 6: 989-95 (1993); Desplancq D etal., Protein Eng 7: 1027-33 (1994); Whitlow M et al., Protein Eng 7,1017-26 (1994); Kortt A et al., Protein Eng 10: 423-33 (1997); Arndt Ket al., Biochemistry 37: 12918-26 (1998); Atwell J et al., Protein Eng12: 597-604 (1999)).

In general, scFv structures with a relatively short linker of five toten amino acid residues or less have a greater propensity forhomo-dimerization (Arndt K et al., Biochemistry 37: 12918-26 (1988);Holliger P et al., Proc Natl Acad Sci USA 90: 6444-8 (1993); Perisic Oet al., Structure 2: 1217-26 (1994); Atwell J et al., Mol Immunol 33:1301-12 (1996); Iliades P et al., FEBS Lett 409: 437-41 (1997); Kortt Aet al., Protein Eng 10: 423-33 (1997); Metzger D et al., Protein Eng 10:423-33 (1997); Pei X et al., Proc Natl Acad Sci USA 94: 9637-42 (1997);Atwell J et al., Protein Eng 12: 597-604 (1999): Denton G et al., CancerImmunol Immunother 48: 29-38 (1999); Le Gall F et al., FEBS Lett 453:164-8 (1999); Atwell J et al., Protein Eng 12: 597-604 (1999); Dolezal Oet al., Protein Eng 13: 565-74 (2000); Nielsen U et al., Cancer Res 60:6434-40 (2000); Todorovska A et al., J Immunol Methods 248: 47-66(2001): Wu A et al., Protein Eng 14: 1025-33 (2001): Arndt M et al.,FEBS Lett 578: 257-61 (2004); Le Gall F et al., J Immunol Methods 285:111-27 (2004)). In contrast, scFvs with linkers comprising at least 12amino acid residues predominantly form monomers with only a minorityfraction undergoing spontaneous multimerization (Nielsen U et al.,Cancer Res 60: 6434-40 (2000); Denton G et al., Cancer ImmunolImmunother 48: 29-38 (1999): Kortt A et al., Biomol Eng 18: 95-108(2001): Volkel T et al., Protein Eng 14: 815-23 (2001)).

The use of linkers of three amino acid residues or fewer may promotemultimerization to higher order structures larger than dimeric forms. Ifan scFv has a linker of less than 3 residues, then trimerization may befavored (Iliades P et al., FEBS Lett 409: 437-41 (1997)); Kortt A etal., Biomol Eng 18: 95-108 (2001); Todorovska A et al., J ImmunolMethods 248: 47-66 (2001): Arndt M et al., FEBS Lett 578: 257-61(2004)). Furthermore, scFvs with very short linkers, e.g., linkers of 2amino acid residues or less, often form trimers and/or mixtures oftrimers and tetramers (Pei X et al., Proc Natl Acad Sci USA 94: 9637-42(1997); Hudson P, Kortt A, J Immunol Methods 231: 177-89 (1999); DolezalO et al., Protein Eng 13: 565-74 (2000): Power B et al., Protein Sci 12:734-47 (2003): Le Gall F et al., J Immunol Methods 285: 111-27 (2004)).In certain arrangements with short linkers, tetramers may be favored(Dolezal O et al., Protein Eng 13: 565-74 (2003): Arndt M et al., FEBSLett 578: 257-61 (2004)). Multimeric structures can be formed by scFvslacking any linker, i.e. having a linker length of zero amino acidresidues. For example, the direct linkage of variable domains with V_(L)before V_(H) may favor the formation of tetrabodies (Iliades P et al.,FEBS Lett 409: 437-41 (1997)) whereas V_(H)before V_(L) may favortrimers (Kortt A et al., Protein Eng 10: 423-33 (1997)).

In addition to the linker length, the orientation of the variabledomains may affect multimerization characteristics (Huston J et al.,Proc Natl Acad Sci USA 85, 5879-83 (1988); Padlan E, Mol Immunol 31:169-217 (1994); Kortt A et al., Protein Eng 10: 423-33 (1997); Dolezal,O et al., Protein Eng 13: 565-74 (2000); Carmichael J et al., J Mol Biol326: 341-51 (2003): Arndt M et al., FEBS Lett 578: 257-61 (2004)). Ithas been suggested that the V_(L)-V_(H) orientation exhibits a greatertendency to form higher molecular weight oligomers than does the reverseorientation because the V_(L)-V_(H) orientation is more constrained(Kortt A et al., Protein Eng 10: 423-33 (1997); Dolezal, O et al.,Protein Eng 13: 565-74 (2000); Plückthun A, Pack P, Immunotechnology 3:83-105 (1997)).

The same linker has shown variability in its effect on scFvmultimerization depending on the V_(H) and V_(L) orientation, such as,e.g., affecting the relative proportions of dimeric to trimeric forms(Le Gall F et al., FEBS Lett 453: 164-8 (1999): Arndt M et al., FEBSLett 578: 257-61 (2004); Le Gall F et al., J Immunol Methods 285: 111-27(2004)).

Camelid V_(H)H immunoglobulin domains have been multimerized usingparticular hinges and covalently linked multi V_(H)H chains (tandem)(Fraile S et al., Mol Microbiol 53: 1109-21 (2004); Zhang J et al., JMol Biol 335: 49-56 (2004)). Immunoglobulin domains from Chondrichthyes,such as IgNARs, have been multimerized using certain hinges orcysteine-mediated disulfide bond stabilization (see e.g. Simmons et al.,J Immunol Methods 315: 171-84 (2006)).

Thus, the generation of multivalent cell-targeting molecules comprisingvarious immunoglobulin domains may be controlled by molecularengineering strategies which are either covalent or non-covalent, suchas, e.g., covalent strategies involving single-chain tandemarrangements, covalent strategies involving cysteine-mediated, disulfidebond stabilized multimers, and/or non-covalent strategies involvingdimerization domains, linker choice, and/or variable domain order.Multiple strategies (e.g., linker-related non-covalent multimerizationand covalent disulfide bond stabilization) may be combined when creatingstructures that are multivalent cell-targeting molecules of the presentinvention (see e.g. Lu D et al., J Immunol Methods 279: 219-32 (2003)).

For certain applications, the stability of the relative proportion ofmultivalent cell-targeting molecule(s) to total cell-targeting moleculesin a composition of the present invention may be important to thecomposition's effectiveness. For example in certain medicalapplications, the stability of the relative proportions of multivalentcell-targeting molecule(s) of the present invention to monovalentcell-targeting molecule(s) may be important. In certain applications,the stability of the relative proportions of bivalent cell-targetingmolecules to higher-valence cell-targeting molecules may be important.In certain applications the stability of the relative proportion ofbivalent cell-targeting molecules to non-bivalent cell-targetingmolecules may be important.

For certain embodiments, a one or more steps of controlledmultimerization of some or all of the components of a multivalentcell-targeting molecule of the present invention may be used to producea composition of the present invention.

For certain applications, the minimization or otherwise controlling ofunwanted aggregation and/or multimerization of cell-targeting moleculesmay be important for certain compositions of the present invention. Forexample with certain proteinaceous therapeutics, the aggregation and/ormultimerization of the therapeutic molecule can in certain situationsincrease the risk for unwanted immune responses in recipients of theproteinaceous therapeutic. In particular, cell-targeting moleculeaggregation and/or multimerization to higher molecular weight complexesmay increase the risk of unwanted immune responses after administrationof certain cell-targeting molecule compositions to certain recipients.In addition, misfolded proteins and degraded protein products canexhibit increased immunogenicity as compared to their properly foldedcounterparts.

For all of these reasons and depending on the specific application, theskilled worker will appreciate whether there is a need to consider 1)the stability of multivalent cell-targeting molecules of thecompositions of the present invention and 2) the stability of the ratiosof different cell-targeting molecules present in compositions of thepresent invention. For example, in certain embodiments, the multivalentcell-targeting molecule of the present invention and compositionsthereof are the result of controlled multimerization and/or certainpurification steps. Similarly, in certain embodiments, the multivalentcell-targeting molecule of the present invention will be engineered toeliminate or reduce certain multimerization possibilities. In certainembodiments, the multivalent cell-targeting molecule of the presentinvention will be designed to avoid the formation of unwantedaggregates, such as, e.g., under certain storage conditions like in anaqueous solution at 8, 4, 2, −4, −10, −20, or −25° C.

For certain applications of the compositions of the present invention,it may be desirable to minimize in the composition of the presentinvention the amount of: 1) high molecular weight, multivalentcell-targeting molecules (e.g. molecules greater than 175, 180, 190,200, or 250 kDa or larger): 2) greatly multivalent cell-targetingmolecules (i.e. molecules comprising five or more cell-targeting bindingregions); 3) multimers of cell-targeting molecules which are highmolecular weight, multivalent cell-targeting molecules representing #1and/or greatly multivalent cell-targeting molecules representing #2(e.g. certain, large, noncovalent multimers of cell-targetingmolecules): 3) misfolded proteins (e.g., misfolded cell-targetingproteins or protein components thereof); and/or 4) degradation products(e.g. unwanted protein fragments of a proteinaceous component of amultivalent cell-targeting molecule, such as, e.g., a polypeptidefragment of a Shiga toxin effector region or cell-targeting bindingregion). For example, a rationale to minimize the amount of any of thetypes of molecules listed as #1-#4 above might be for medicalapplications where the presence of a certain amounts of these moleculesmight increase the potential for unwanted antigenic and/or immunogenicreactions in a recipient of a compositions of the present invention,such as, e.g., by the presence of these molecules revealing new epitopesor by forming repetitive motifs more readily identified by a recipient'simmune system as foreign.

The skilled worker may use routine methods to assess multimerizationstates of the multivalent cell-targeting molecules of the presentinvention and/or molecules present in the compositions of the presentinvention. The skilled worker may use routine methods to minimize thepresence or relative proportion of cell-targeting molecule aggregates,high molecular weight cell-targeting protein multimers, misfoldedcell-targeting proteins, and degradation products of cell-targetingprotein in the compositions of the present invention.

In certain embodiments of the compositions of the present invention, therelative proportion of bivalent, trivalent, and/or tetravalent forms ofmultivalent cell-targeting molecule(s) is maximized, such as by furtherpurifying away from monovalent cell-targeting protein(s), highermolecular weight cell-targeting molecule(s), misfolded cell-targetingprotein(s), and/or protein degradation product(s).

The skilled worker may use routine methods to create a multivalentcell-targeting molecule of the present invention, and compositionsthereof. The skilled worker may use routine methods to stabilize therelative proportions of certain multivalent cell-targeting molecules toother molecules in a composition of the present invention, including theproportions of different multimeric forms of cell-targeting molecules,such as, e.g., the proportions of covalently linked, multimeric,multivalent cell-targeting molecules to non-covalently linked,multimeric, multivalent cell-targeting molecules (see e.g. Gil D, SchrumA, Adv Biosci Biotechnol 4: 73-84 (2013): WO2005000898). For example,the multimerization of cell-targeting molecule(s) in compositions of thepresent invention may be controlled and/or minimized, such as, e.g., bychoosing certain linkers to link and/or associate different componentsand/or subunits of the cell-targeting molecule(s) present in thecompositions of the present invention. For example, in certainembodiments, the cell-targeting binding region of the multivalentcell-targeting molecule of the present invention is engineered tominimize the formation of unwanted, intermolecular associations,multimers, and/or aggregates, such as, e.g., by usingdisulfide-stabilized scFvs, Fv fragments, or Fabs (see e.g. Reiter Y etal., J Biol Chem 269: 18327-31 (1994): Kuan C, Pastan I, Biochemistry35: 2872-7 (1996); Almog O et al., Proteins 31: 128-38 (1998);Schoonjans R et al., J Immunol 165: 7050-7 (2000): Olafsen T et al.,Protein Eng Des Sel 17: 21-7 (2004): Gil D, Schrum A, Adv BiosciBiotechnol 4: 73-84 (2013); U.S. 20120283418); base loop connections(see e.g. Brinkmann U et al., J Mol Biol 268: 107-17 (1997)); and/orother modifications, such as the addition of charged resides, glycans,and/or immunoglobulin-domain truncations (see e.g. Gong R et al., MolPharm 10: 2642-52 (2013); Lee C et al., Trends Biotechnol 31: 612-20(2013)).

In certain embodiments of the present invention, the multivalentcell-targeting molecule of the present invention comprises acell-targeting binding region which is an scFv engineered not toaggregate, such as, e.g., by using a shorter linker (typically less thantwelve amino acid residues) and/or disulfide-stabilized linker thatlinks the heavy and light chain regions of the scFv (see e.g., BrinkmannU et al., Proc Natl Acad Sci USA 90: 7538-42 (1993); Whitlow M et al.,Protein Engineering 6: 989-95 (1993); Reiter Y et al., Biochemistry 33:5451-9 (1994); Gong R et al., Molecular Pharmaceutics 10: 2642-52(2013)).

In certain embodiments, the multivalent cell-targeting moleculecomposition of the present invention minimizes the proportion relativeto other cell-targeting molecules of certain, multivalent cell-targetingmolecule(s) with a valence greater than two. In certain embodiments, themultivalent cell-targeting molecule composition of the present inventioncomprises a relative percentage of multivalent cell-targeting moleculeswith a valence of greater than four which is 15%, 10%, 7.5%, 5%, 2%, 1%,or less of the total cell-targeting molecules in the composition.

In certain embodiments, a multivalent cell-targeting moleculecomposition of the present invention comprises a relative percentage ofcell-targeting molecules with a valence of greater than three to othercell-targeting molecules which is 15%, 10%, 7.5%, 5%, 2%, 1%, or less ofthe total cell-targeting molecules in the composition.

In certain embodiments, a multivalent cell-targeting moleculecomposition of the present invention comprises a percentage ofcell-targeting molecules with a valence greater than two which is 15%,10%, 7.5%, 50%, 2%, 1%, or less of the total cell-targeting molecules inthe composition.

In certain embodiments, the composition of the present inventionmaximizes the relative proportion of multivalent cell-targetingmolecule(s) with exactly two cell-targeting binding regions to totalcell-targeting molecules. Thus, in certain embodiments, a composition ofthe present invention comprises a proportion of cell-targeting moleculewith only two cell-targeting binding regions which is 80%, 85%, 88%,90%, 92%, 93%, or more of the total cell-targeting molecules in thecomposition.

For certain applications, it may be desirable to maintain stability(e.g., the stability of associations and/or linkages between componentsand/or subunits of the multivalent cell-targeting molecules) ofmultivalent cell-targeting molecule(s) in a multivalent composition ofthe present invention, such as, e.g., to minimize degradation duringformulation, storage (such as, e.g., storage in an aqueous solution at8, 4, 2, −4, −10, −20, or −25° C.), and/or after administration to arecipient. The skilled worker may use well known methods to minimizecomponent or subunit separation for a multivalent cell-targetingmolecule of the present invention, such as, e.g., by usinghigh-stability linkages between the Shiga toxin effector polypeptide(s)and binding region(s) and/or by engineering disulfide linkages betweencomponents, regions, or sub-regions of a multivalent cell-targetingmolecule or between monovalent cell-targeting proteins to generatemultivalent cell-targeting protein(s) of the present invention (see e.g.Gil D. Schrum A. Adv Biosci Biotechnol 4: 73-84 (2013)). The skilledworker may use the addition or maintenance of intermolecular disulfidebonds to stabilize certain cell-targeting binding regions of themultivalent cell-targeting molecules of the present invention (see e.g.Glockshuber R et al., Biochemistry 29: 1362-7 (1990); Stanfield R etal., Science 305: 1770-3 (2004); Hagihara Y et al., J Biol Chem 282:36489-95 (2007): Chan P et al., Biochemistry 47: 11041-54 (2008);Saerens D et al., J Mol Biol 478-88 (2008); Hussack G et al., PLoS One6: e28218 (2011); Govaert J et al., J Biol Chem 287: 1970-9 (2012); KimD et al., Protein Eng Des Sel 25: 581-9 (2012); Gil D, Schrum A, AdvBosci Biotechnol 4: 73-84 (2013); McConnell A et al., Protein Eng DesSel 25: 581-9 (2013); Feige M et al., Proc Natl Acad Sci USA 111:8155-60 (2014); Hagihara Y, Saerens D, Biochim Biophys Acta 1844:2016-2023 (2014): Kim D et al., Mabs 6: 219-35 (2014)).

In certain embodiments, the multivalent cell-targeting molecule of thepresent invention comprises a cell-targeting binding region(s) whichcomprises an immunoglobulin domain and/or Ig-fold structure having anintra-domain disulfide bond, such as, e.g., the disulfide bond foundnatively between the B and F β strands of certain immunoglobulins and/ora disulfide bond between their heavy and light chains of or derived froman immunoglobulin. However, in certain embodiments of the multivalentcell-targeting molecules of the present invention, the molecules arevery stable even though they do not comprise an intra-domain disulfidebond or any intra-domain disulfide bond within one or morecell-targeting binding regions (see e.g. Proba K et al., Biochemistry37: 13120-7 (1998): W6m A. Plückthun A, Biochemistry 37: 13120-7 (1998):Wöm A, Plückthun A, FEBS Lett 427: 357-61 (1998): Ramm K et al., J MolBiol 290: 535-46 (1999); Tanaka T, Rabbitts T, J Mol Biol 376: 749-57(2008)).

In certain embodiments, the composition of the present inventioncomprises a multivalent cell-targeting molecule with one or moredisulfide bonds between two or more cysteine residues contained withinShiga toxin effector polypeptide regions of different polypeptidechains. In certain embodiments, the composition of the present inventioncomprises a proteinaceous, dimeric, multivalent cell-targeting moleculewith five disulfide bonds, such as, e.g., the dimeric, multivalentcell-targeting molecule comprising: 1) four, intramolecular, disulfidebonds representing two disulfide bonds per immunoglobulin-derivedcell-targeting binding region and where each disulfide bond involves apair of cysteine residues and wherein one cysteine residue of each pairis within an immunoglobulin heavy chain derived domain and the othercysteine residue of the pair is within an immunoglobulin light chainderived domain: and 2) one, intermolecular, disulfide bond bridging two,Shiga toxin effector regions wherein the disulfide bond occurs between apair of cysteine residues where each cysteine residue of the pair iswithin a Shiga toxin effector region but the Shiga toxin effectorregions are within different polypeptide chains representing differentsubunits of a multivalent cell-targeting protein of the presentinvention.

Certain embodiments of the multivalent cell-targeting molecules of thepresent invention are cytotoxic, cell-targeting, fusion proteins.Certain further embodiments are the cell-targeting molecules whichcomprise or consist essentially of two or more of the polypeptides shownin SEQ ID NOs: 252-255, 259-278, and 288-748.

For the purposes of the present invention, the specific order ororientation is not fixed for the Shiga toxin effector polypeptide(s) andthe two or more binding regions in relation to each other or the entiremultivalent cell-targeting molecule of the present invention. Thecomponents of the multivalent cell-targeting molecules of the presentinvention may be arranged in any order provided that the desiredactivities of the binding regions and the Shiga toxin effectorpolypeptide(s) are not eliminated, notably the ability of thecell-targeting molecule to bind target-expressing cells, the ability ofthe cell-targeting molecule to internalize into a target cell, and theability of the Shiga toxin effector polypeptide component to deliver aCD8+ T-cell epitope-peptide cargo to the MHC class I pathway of a cellin which it is present. Other desired activities include providing themultivalent cell-targeting molecule with the ability to, e.g., rapidlyinduce cellular internalization; cause efficient internalization:intracellularly route to a desired subcellular compartment(s): causecytostasis; cause cytotoxicity: selectively kill target-expressingcells: deliver exogenous materials into the interior of a cell;diagnosis a disease, disorder, or condition; and/or treat a disease,disorder, or condition in a patient in need thereof.

Cell-targeting molecules of the present invention each comprise acell-targeting binding region which can bind specifically to at leastone extracellular target biomolecule in physical association with acell, such as a target biomolecule expressed on the surface of a cell.This general structure is modular in that any number of diversecell-targeting moieties may be used as a binding region of acell-targeting molecule of the present invention. It is within the scopeof the present invention to use fragments, variants, and/or derivativesof the cell-targeting molecules of the present invention which contain afunctional binding site to any extracellular part of a targetbiomolecule, and even more preferably capable of binding a targetbiomolecule with high affinity (e.g. as shown by a K_(D) less than 10⁻⁹moles/liter). For example, while the invention provides polypeptidesequences that can bind to human proteins, any binding region that bindsan extracellular part of a target biomolecule with a dissociationconstant (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter, preferably less than 200nM, may be substituted for use in making cell-targeting molecules of theinvention and methods of the invention.

III. General Functions of the Cell-Targeting Molecules of the PresentInvention

The cell-targeting molecules of the present invention may be used ascell-targeted cytotoxic molecules, therapeutic molecules, cell-labelingmolecules, and diagnostic molecules, e.g., via their abilities to targetspecific cell-types based on cell-surface marker expression, enteringtarget cells, delivering intracellularly its heterologous CD8+ T-cellepitope-peptide cargo to the MHC class I pathway resulting incell-surface presentation of the CD8+ T-cell epitope-peptide by thetarget cell(s).

For certain embodiments, the cell-targeting molecule of the presentinvention provides, after administration to a chordate, one or more ofthe following: 1) potent and selective killing of targeted cells, e.g.,infected and/or neoplastic cells, 2) linkage stability between thecell-targeting binding region and the Shiga toxin effector polypeptidecomponent while the cell-targeting molecule is present in extracellularspaces, e.g., in the circulatory system of a chordate (see e.g. WO2015/1917(A), 3) low levels of off-target cell deaths and/or unwantedtissue damage (see e.g. WO 2015/191764), and 4) cell-targeted deliveryof a heterologous, CD8+ T-cell epitope cargo for cell-surfacepresentation by MHC class I molecules of target cells in order tostimulate desirable immune responses, such as, e.g., the recruitment ofCD8+ CTLs and the localized release of immuno-stimulatory cytokines at atissue locus, e.g. a tumor mass. Furthermore, the presentation ofdelivered, heterologous, CD8+ T-cell epitope-peptides by target cellsmarks those presenting cells with pMHC Is that can be detected for thepurposes of gathering information, such as, e.g., for diagnosticinformation.

The cell-targeting molecules of the present invention are useful indiverse applications involving, e.g., targeted delivery of a CD8+ T-cellepitope-cargo, immune response stimulation, targeted cell-killing,targeted cell growth inhibition, biological information gathering,and/or remediation of a health condition. The cell-targeting moleculesof the present invention are useful as therapeutic and/or diagnosticmolecules, such as, e.g., as cell-targeting, nontoxic, deliveryvehicles: cell-targeting, cytotoxic, therapeutic molecules; and/orcell-targeting, diagnostic molecules; for examples in applicationsinvolving the in vivo targeting of specific cell-types for the diagnosisor treatment of a variety of diseases, including cancers, immunedisorders, and microbial infections. Certain cell-targeting molecules ofthe present invention may be used to treat a chordate afflicted with atumor or cancer by enhancing the effectiveness of that chordate'santi-tumor immunity, particularly involving CD8+ T-cell mediatedmechanisms (see e.g. Ostrand-Rosenberg S, Curr Opin Immunol 6: 722-7(1994); Pietersz G et al., Cell Mol Life Sci 57: 290-310 (2000); LazouraE et al., Immunology 119: 306-16 (2006)).

Depending on the embodiment, a cell-targeting molecule of the presentinvention may have or provide one or more of the followingcharacteristics or functionalities: (1) in vivo stimulation of CD8+T-cell immune response(s). (2) de-immunization (see e.g. WO 2015/113005,WO 2015/113007, and WO 2015/191764), (3) protease-cleavage resistance(see e.g. WO 2015/191764 and WO 2015/191764), (4) potent cytotoxicity atcertain concentrations, (5) selective cytotoxicity, (6) low off-targettoxicity in multicellular organisms at certain doses or dosages (seee.g. WO 2015/191764 and WO 2015/191764), and/or (7) intracellulardelivery of a cargo consisting of an additional material (e.g. a nucleicacid or detection promoting agent). Certain embodiments of thecell-targeting molecules of the present invention are multi-functionalbecause the molecules have two or more of the characteristics orfunctionalities described herein. Certain further embodiments of thecell-targeting molecules of the present invention provide all of theaforementioned characteristics and functionalities in a single molecule.

The mechanisms of action of the therapeutic, cell-targeting molecules ofthe present invention include direct target cell-killing via Shiga toxineffector functions, indirect cell-killing via intercellularimmune-cell-mediated processes, and/or educating a recipient's immunesystem to reject certain cells and tissue loci, e.g. a tumor mass, as aresult of “CD8+ T-cell epitope seeding.”

A. Delivery of the Heterologous, CD8+ T-Cell Epitope Cargo to the MHCClass I Presentation Pathway of a Target Cell

One of the primary functions of the cell-targeting molecules of thepresent invention is cell-targeted delivery of one or more heterologous,CD8+ T-cell epitope-peptides for MHC class I presentation by a chordatecell. The cell-targeting molecules of the present invention are modularscaffolds for use as general delivery vehicles of virtually any CD8+T-cell epitope cargo to virtually any chordate target cell. Targeteddelivery requires the cell-targeting molecule to specifically bind to acertain target cell, enter the target cell, and deliver an intactheterologous, CD8+ T-cell epitope-peptide cargo to a subcellularcompartment competent for entry into the MHC class I presentationpathway. Delivery of a CD8+ T-cell epitope-peptide cargo to the MHCclass I presentation pathway of a target cell using a cell-targetingmolecule of the invention can be used to induce the target cell topresent the epitope-peptide in association with MHC class I molecules ona cell surface.

By using immunogenic MHC class I epitopes, such as, e.g., from a knownviral antigen, as heterologous, CD8+ T-cell epitope-peptide cargos ofthe cell-targeting molecules of the present invention, the targeteddelivery and presentation of immuno-stimulatory antigens may beaccomplished in order to stimulate a beneficial function(s) of achordate immune cell, e.g. in vitro, and/or a chordate immune system invivo.

In a chordate, the presentation of an immunogenic, CD8+ T-cell epitopeby the MHC class I complex can target the presenting cell for killing byCTL-mediated cytolysis, promote immune cells into altering themicroenvironment, and signal for the recruitment of more immune cells tothe target cell site within the chordate.

Certain cell-targeting molecules of the present invention are capable ofdelivering under physiological conditions its heterologous, CD8+ T-cellepitope-peptide cargo to the MHC class I pathway of a target chordatecell for presentation of the delivered T-cell epitope complexed with aMHC class I molecule. This may be accomplished by exogenousadministration of the cell-targeting molecule into an extracellularspace, such as, e.g., the lumen of a blood vessel, and then allowing forthe cell-targeting molecule to find a target cell, enter the cell, andintracellularly deliver its CD8+ T-cell epitope cargo. The presentationof a CD8+ T-cell epitope by a target cell within a chordate can lead toan immune response(s), including responses directly to the target celland/or general responses in the tissue locale of the target cell withinthe chordate.

The applications of these CD8+ T-cell epitope cargo delivery and MHCclass I presenting functions of the cell-targeting molecules of thepresent invention are vast. For example, the delivery of a CD8+ epitopeto a cell and the MHC class I presentation of the delivered epitope bythe cell in a chordate can cause the intercellular engagement of a CD8+effector T-cell and may lead to a CTL(s) killing the target cell and/orsecreting immuno-stimulatory cytokines.

The cell-targeting molecules of the present invention are capable, uponexogenous administration, of delivering one or more CD8+ T-cell epitopecargos for MHC class I presentation by a nucleated, chordate cell. Forcertain embodiments, the cell-targeting molecules of the presentinvention are capable of binding extracellular target biomoleculesassociated with the cell surface of particular cell-types and enteringthose cells. Once internalized within a targeted cell-type, thecell-targeting molecules of the invention are capable of delivering aCD8+ T-cell epitope cargo to the MHC class I presentation pathway andcertain further embodiments of the cell-targeting molecules of theinvention are capable of routing a Shiga toxin effector polypeptidecomponent (whether catalytically active, reduced-cytotoxicity, ornon-toxic) to the cytosol of the cell.

For certain embodiments, the cell-targeting molecule of the presentinvention is capable, from an extracellular space, of delivering one ormore heterologous, CD8+ T-cell epitope-peptide cargos to the proteasomeof a target cell. The delivered CD8+ T-cell epitope-peptide cargo canthen be proteolytic processed and presented by the MHC class I pathwayon the surface of the target cell. For certain embodiments, thecell-targeting molecule of the present invention is capable ofdelivering the heterologous, CD8+ T-cell epitope-peptide cargo, which isassociated with the cell-targeting molecule, to a MHC class I moleculeof a cell for presentation of the epitope-peptide by the MHC class Imolecule on a surface of the cell. For certain embodiments, uponcontacting a cell with the cell-targeting molecule of the presentinvention, the cell-targeting molecule is capable of delivering theheterologous, CD8+ T-cell epitope-peptide cargo, which is associatedwith the cell-targeting molecule, to a MHC class I molecule of the cellfor presentation of the epitope-peptide by the MHC class I molecule on asurface of the cell.

For certain embodiments, the cell-targeting molecule of the presentinvention is capable, upon administration to a chordate subject, oftargeting delivery of one or more heterologous, CD8+ T-cell epitopecargos for MHC class I presentation by specific target cells within thesubject.

In principle, any CD8+ T-cell epitope-peptide may be chosen for use in acell-targeting molecule of the present invention. Thus, cell-targetingmolecules of the invention are useful for labeling the surfaces oftarget cells with MHC class I molecules complexed with theepitope-peptide of your choice.

Every nucleated cell in a mammalian organism may be capable of MHC classI pathway presentation of immunogenic, CD8+ T-cell epitope peptides ontheir cell outer surfaces complexed to MHC class I molecules. Inaddition, the sensitivity of T-cell epitope recognition is so exquisitethat only a few MHC-I peptide complexes are required to be presented toresult in an immune response, e.g., even presentation of a singlecomplex can be sufficient for the intercellular engagement of a CD8+effector T-cell (Sykulev Y et al., Immunity 4: 565-71 (1996)). Targetcells of a cell-targeting molecule of the present invention can bevirtually any nucleated chordate cell-type and need not be immune cellsand/or professional antigen presenting cells. Examples of professionalantigen presenting cells include dendritic cells, macrophages, andspecialized epithelial cells with functional MHC class II systems. Infact, preferred embodiments of the cell-targeting molecules of thepresent invention do not target professional antigen presenting cells.One reason is that an undesirable immune response as a result of theadministration of the cell-targeting molecule of the present inventionwould be a humoral response directed to the cell-targeting moleculeitself, such as, e.g., an anti-cell-targeting molecule antibodyrecognizing an epitope in the cell-targeting molecule. Thus,professional antigen presenting cells and certain immune cell-types arenot to be targeted by certain embodiments of the cell-targetingmolecules of the present invention because the uptake of thecell-targeting molecule of the present invention by these cells may leadto the recognition of CD4+ T-cell and B-cell epitopes present in thecell-targeting molecule, particularly in the Shiga toxin effectorpolypeptide component(s) and/or an antigenic cargo, but also includingin the binding region.

The ability to deliver a CD8+ T-cell epitope by certain embodiments ofthe cell-targeting molecules of the present invention may beaccomplished under varied conditions and in the presence of non-targetedbystander cells, such as, e.g., an ex vivo manipulated target cell, atarget cell cultured in vitro, a target cell within a tissue samplecultured in vitro, or a target cell in an in vivo setting like within amulticellular organism.

In order for a cell-targeting molecule of the present invention tofunction as designed, the cell-targeting molecule must 1) enter a targetcell and 2) localize its CD8+ T-cell epitope-peptide cargo to asubcellular location competent for entry into the MHC class I pathway.Commonly, cell-targeting molecules of the invention accomplish targetcell internalization via endocytosis, such as, e.g., due to a naturalprocess involving the extracellular target biomolecule bound by thecell-targeting molecule. Once the cell-targeting molecule of theinvention is internalized, it will typical reside in an early endosomalcompartment, such as, e.g., endocytotic vesicle and be destined fordestruction in a lysosome or late endosome. A cell-targeting moleculemust avoid complete sequestration and degradation such that at least aportion of the cell-target molecule comprising the T-cellepitope-peptide cargo escapes to another subcellular compartment.Furthermore, the target cell should either express a MHC class Imolecule or be capable of being induced to express a MHC class Imolecule.

The expression of the MHC class I molecule need not be native in orderfor cell-surface presentation of a heterologous, CD8+ T-cellepitope-peptide (delivered as a cargo by a cell-targeting molecule ofthe present invention) complexed with a MHC class I molecule. Forcertain embodiments of the present invention, the target cell may beinduced to express MHC class I molecule(s) using a method know n to theskilled worker, such as, e.g., by treatment with IFN-γ.

Commonly, cell-targeting molecules of the invention accomplish MHC classI pathway delivery by localizing their CD8+ T-cell epitope-peptidecargos to proteasomes in cytosolic compartments of target cells.However, for certain embodiments, the cell-targeting molecule of thepresent invention may deliver a heterologous, CD8+ epitope-peptide tothe MHC class I presentation pathway without the epitope-peptide everentering a cytosolic compartment and/or without the epitope-peptide everbeing proteolytically processed by the proteasome.

For certain embodiments of the present invention, the target cell may beinduced to express different proteasome subunits and/or proteasomesubtypes using a method know n to the skilled worker, such as, e.g., bytreatment with IFN-γ and/or TNF-α. This can alter the positioning and/orrelative efficiency of proteolytic processing of CD8+ epitope peptidesdelivered into the cell, such as, e.g., by altering the relative levelsof peptidase activities of proteasomes and proteasome subtypes.

The CD8+ T-cell epitope delivering functions of the cell-targetingmolecules of the present invention can be detected and monitored by avariety of standard methods known in the art to the skilled workerand/or described herein. For example, the ability of cell-targetingmolecules of the present invention to deliver a CD8+ T-cellepitope-peptide cargo and drive presentation of this peptide by the MHCclass I system of target cells may be investigated using various invitro and in vivo assays, including, e.g., the directdetection/visualization of MHC class I/peptide complexes (pMHC Is),measurement of binding affinities for the T-cell peptide to MHC class Imolecules, and/or measurement of functional consequences of pMHC Ipresentation on target cells, e.g., by monitoring cytotoxic T-lymphocyte(CTL) responses (see e.g. Examples, infra).

Certain assays to monitor and quantitate the CD8+ T-cell epitope cargodelivering function of the cell-targeting molecules of the presentinvention involve the direct detection of a specific pMHC Is in vitro orex vivo. Common methods for direct visualization and quantitation ofpMHC Is involve various immuno-detection reagents known to the skilledworker. For example, specific monoclonal antibodies can be developed torecognize a particular pMHC I. Similarly, soluble, multimeric T cellreceptors, such as the TCR-STAR reagents (Altor Bioscience Corp.,Miramar, F L, U.S.) can be used to directly visualize or quantitatespecific pMHC Is (Zhu X et al., J Immunol 176: 3223-32 (2006) see e.g.,Examples, infra). These specific mAbs or soluble, multimeric T-cellreceptors may be used with various detection methods, including, e.g.immunohistochemistry, flow cytometry, and enzyme-linked immunosorbentassay (ELISA).

An alternative method for direct identification and quantification ofpMHCs involves mass spectrometry analyses, such as, e.g., the ProPresentAntigen Presentation Assay (Prolmmune, Inc., Sarasota, Fla., U.S.) inwhich peptide-MHC class I complexes are extracted from the surfaces ofcells, then the peptides are purified and identified by sequencing massspectrometry (Falk K et al., Nature 351: 290-6 (1991)).

In certain assays to monitor the CD8+ T-cell epitope delivery and MHCclass I presentation function of the cell-targeting molecules of thepresent invention involve computational and/or experimental methods tomonitor MHC class I and peptide binding and stability. Several softwareprograms are available for use by the skilled worker for predicting thebinding responses of peptides to MHC class I alleles, such as, e.g., TheImmune Epitope Database and Analysis Resource (IEDB) Analysis ResourceMHC-I binding prediction Consensus tool (Kim Y et al., Nucleic Acid Res40: W525-30 (2012)). Several experimental assays have been routinelyapplied, such as, e.g., cell surface binding assays and/or surfaceplasmon resonance assays to quantify and/or compare binding kinetics(Miles K et al., Mol Immunol 48: 728-32 (2011)).

Alternatively, measurements of the consequence of pMHC I presentation onthe cell surface can be performed by monitoring the cytotoxic Tlymphocyte (CTL) response to the specific complex. These measurements byinclude direct labeling of the CTLs with MHC class I tetramer orpentamer reagents. Tetramers or pentamers bind directly to T cellreceptors of a particular specificity, determined by the MajorHistocompatibility Complex (MHC) allele and peptide complex.Additionally, the quantification of released cytokines, such asinterferon gamma or interleukins by ELISA or enzyme-linked immunospot(ELIspot) is commonly assayed to identify specific CTL responses. Thecytotoxic capacity of CTL can be measured using a number of assays,including the classical 51 Chromium (Cr) release assay or alternativenon-radioactive cytotoxicity assays (e.g., CytoTox96% non-radioactivekits and CellTox™ CellTiter-GLOX kits available from Promega Corp.,Madison, Wis., U.S.). Granzyme B ELISpot, Caspase Activity Assays orLAMP-1 translocation flow cytometric assays. To specifically monitor thekilling of target cells, carboxyfluorescein diacetate succinimidyl ester(CFSE) can be used to easily and quickly label a cell population ofinterest for in vitro or in vivo investigation to monitor killingofepitope specific CSFE labeled target cells (Durward M et al., J VisExp 45 pii 2250 (2010)).

In vivo responses to MHC class I presentation can be followed byadministering a MHC class I/antigen promoting agent (e.g., a peptide,protein or inactivated/attenuated virus vaccine) followed by challengewith an active agent (e.g. a virus) and monitoring responses to thatagent, typically in comparison with unvaccinated controls. Ex vivosamples can be monitored for CTL activity with methods similar to thosedescribed previously (e.g. CTL cytotoxicity assays and quantification ofcytokine release).

MHC class I presentation in an organism can be followed by reverseimmunology. For example, HLA-A, HLA-B, and/or HLA-C molecule complexesare isolated from cells intoxicated with a cell-targeting molecule ofthe present invention comprising antigen X after lysis using immuneaffinity (e.g., an anti-MHC I antibody “pulldown” purification) andassociated peptides (i.e., the peptides that were bound by the isolatedpMHC Is) are recovered from the purified complexes. The recoveredpeptides are analyzed by sequencing mass spectrometry. The massspectrometry data is compared against a protein database libraryconsisting of the sequence of the exogenous (non-self) peptide (antigenX) and the international protein index for humans (representing “self”or non-immunogenic peptides). The peptides are ranked by significanceaccording to a probability database. The detected antigenic (non-self)peptide sequences are listed. The data is verified by searching againsta scrambled decoy database to reduce false hits (see e.g. Ma B, JohnsonR, Mol Cell Proteomics 11: O111.014902 (2012)). The results candemonstrate which peptides from the CD8+ T-cell antigen X are presentedin MHC I complexes on the surface of cell-targeting molecule intoxicatedtarget cells.

B. Cell Kill: Directly Targeted Shiga Toxin Cytotoxicity and/orIndirectly Targeted Cell-Mediated Cytotoxicity via the Recruitment ofCTLs

Cell-targeting molecules of the present invention can provide cell-typespecific delivery of: 1) CD8+ T-cell epitope cargos to the MHC class Ipresentation pathway for presentation and intercellular engagement ofCTL(s) as well as 2) potent Shiga toxin cytotoxicity to the cytosol.These multiple cytotoxic mechanisms may complement each other, such asby providing both direct (e.g. Shiga toxin catalysis mediated)target-cell-killing and indirect (e.g. CTL-mediated)target-cell-killing.

For certain embodiments, the cell-targeting molecule of the presentinvention is cytotoxic at certain concentrations. The cell-targetingmolecules of the present invention may be used in application involvingindirect (e.g. via intercellular CD8+ immune cell engagement) and/ordirect cell killing mechanisms (e.g. via intracellular toxin effectoractivity). Because Shiga toxins are adapted to killing eukaryotic cells,cytotoxic cell-targeting molecules designed using Shiga toxin A Subunitderived polypeptides can show potent cell-kill activity. Shiga toxin ASubunits and derivatives thereof which comprise active enzymatic domainscan kill a eukaryotic cell once in the cell's cytosol. The fusion of acell-targeting binding region and a heterologous, CD8+ T-cellepitope-peptide to a de-immunized and protease-cleavage resistant, Shigatoxin A Subunit effector polypeptide can be accomplished withoutsignificantly reducing the Shiga toxin effector polypeptide's catalyticand cytotoxic activities (see Examples, infra). Thus, certaincell-targeting molecules of the present invention can provide at leasttwo redundant, mechanisms of target cell killing—(1) indirect, immunecell-mediated killing as a result of heterologous, CD8+ epitope cargodelivery by the cell-targeting molecule of the present invention and (2)direct killing via the functional activity(ies) of a Shiga toxineffector polypeptide component of the cell-targeting molecule of theinvention.

For certain embodiments of the cell-targeting molecules of the presentinvention, upon contacting a target cell physically coupled with anextracellular target biomolecule of the binding region of the molecule,the cell-targeting molecule is capable of causing death of the targetcell. The mechanism of cell-kill may be direct, e.g. via the enzymaticactivity of the Shiga toxin effector polypeptide, or indirect via immunecell-mediated mechanisms, e.g. CTL-mediated target cell cytolysis, andmay be under varied conditions of target cells, such as an ex vivomanipulated target cell, a target cell cultured in vitro, a target cellwithin a tissue sample cultured in vitro, or a target cell in vivo.

The expression of the target biomolecule need not be native in order fortargeted cell killing by a cell-targeting molecule of the invention.Cell-surface expression of the target biomolecule could be the result ofan infection, the presence of a pathogen, and/or the presence of anintracellular microbial pathogen. Expression of a target biomoleculecould be artificial such as, for example, by forced or inducedexpression after infection with a viral expression vector, see e.g.adenoviral, adeno-associated viral, and retroviral systems. An exampleof inducing expression of a target biomolecule is the upregulation ofCD38 expression of cells exposed to retinoids, like all-trans retinoicacid and various synthetic retinoids, or any retinoic acid receptor(RAR) agonist (Drach J et al., Cancer Res 54: 1746-52 (1994); Uruno A etal., J Leukoc Biol 90: 235-47 (2011)). In another example, CD20, HER2,and EGFR expression may be induced by exposing a cell to ionizingradiation (Wattenberg M et al., Br J Cancer 110: 1472-80 (2014)).Further, PSMA expression is upregulated in response to androgendeprivation (see e.g. Chang S et al., Cancer 88: 407-15 (2000): Meller Bet al., EJNMMI Res 5: 66 (2015)).

For certain embodiments of the cell-targeting molecules of the presentinvention, the cell targeting molecules are cytotoxic because deliveryof the molecule's heterologous, CD8+ T-cell epitope(s) cargo results inMHC class I presentation of the delivered epitope-peptide(s) by thetarget cell and immune cell mediated killing of the target cell.

Certain cell-targeting molecules of the present invention may be used inapplications involving indirect cell-kill mechanisms, such as, e.g.,stimulating CD8+ immune cell mediated, target cell killing. Thepresentation by targeted cells of immuno-stimulatory non-self antigens,such as, e.g., known viral epitope-peptides with high immunogenicity,can signal to other immune cells to destroy the target cells and recruitmore immune cells to the target cell site within an organism. Undercertain conditions, the cell-surface presentation of immunogenic CD8+epitope-peptides by the MHC class I complex targets simulates the immunesystem to kill the presenting cell for killing by CD8+ CTL-mediatedcytolysis.

For certain embodiments of the cell-targeting molecules of the presentinvention, upon contacting a cell physically coupled with anextracellular target biomolecule of the molecule's binding region, thecell-targeting molecule is capable of indirectly causing the death ofthe cell, such as, e.g., via the presentation of one or more T-cellepitopes by the target cell and the subsequent recruitment of a CTLs.

In addition, within a chordate, the presentation by target cells of aCD8+ T-cell epitope cargo delivered by the cell-targeting molecule ofthe present invention may provide the additional functionality ofimmuno-stimulation to the local area and/or breaking immuno-tolerance tocertain malignant cells in a local area and/or systemically throughoutthe chordate.

For certain embodiments of the cell-targeting molecules of the presentinvention, upon contacting a cell physically coupled with anextracellular target biomolecule of the binding region, thecell-targeting molecule of the invention is capable of directly causingthe death of the cell, such as, e.g., via the enzymatic activity of aShiga toxin effector polypeptide or a cytotoxic agent described herein.Under certain conditions, certain cell-targeting molecules of thepresent invention are cytotoxic because they comprise a catalyticallyactive, Shiga toxin effector polypeptide component which functions soquickly that it prevents the observation of any functional result ofdelivery of any heterologous, CD8+ T-cell epitope-peptide to the MHCclass I presentation pathway by the cell-targeting molecule; however tobe encompassed within the scope of the claimed cell-targeting molecules,such a cell-targeting molecule must be capable of delivering aheterologous, CD8+ T-cell epitope-peptide cargo from an extracellularspace to the MHC class I presentation pathway of a cell upon exogenousadministration.

In addition, a cytotoxic cell-targeting molecule of the presentinvention that exhibits Shiga toxin effector polypeptide catalyticactivity based cytotoxicity may be engineered by the skilled workerusing routine methods into enzymatically inactive variants to reduce oreliminate Shiga toxin effector based cytotoxicity. The resulting“inactivated” cell-targeting molecule may or may not still be cytotoxicdue to its ability to deliver a heterologous, CD8+ T-cell epitope to theMHC class I system of a target cell and subsequent presentation of thedelivered CD8+ T-cell epitope-peptide by MHC class I molecules on thesurface of the target cell.

For certain embodiments, the Shiga toxin effector polypeptidecomponent(s) of the cell-targeting molecule of the present inventionexhibits low to zero cytotoxicity and thus are referred to herein as“non-cytotoxic and/or reduced cytotoxic.” For certain embodiments, thecell-targeting molecule of the present invention exhibits low to zerocytotoxicity and may be referred to as “non-cytotoxic” and/or “reducedcytotoxic variants.” For example, certain embodiments of thecell-targeting molecules of the present invention do not exhibit asignificant level of Shiga toxin based cytotoxicity wherein at doses ofless than 1,000 nM, 500 nM, 100 nM, 75 nM, 50 nM, there is nosignificant amount of cell death as compared to the appropriatereference molecule, such as, e.g., as measured by an assay known to theskilled worker and/or described herein. For certain further embodiments,the multivalent cell-targeting molecules of the present invention do notexhibit any toxicity at dosages of 1-100 micrograms (pg) per kilogram(kg) of a mammalian recipient. Reduced-cytotoxic variants may still becytotoxic at certain concentrations or dosages but exhibit reducedcytotoxicity, such as, e.g., are not capable of exhibiting a significantlevel of Shiga toxin cytotoxicity in certain situations. Certaincell-targeting molecules of the present invention can be renderednon-cytotoxic or reduced cytotoxic, such as, e.g., via the addition ofone or more amino acid substitutions known to the skilled worker toinactivate a Shiga toxin A Subunit and/or Shiga toxin effectorpolypeptide, including exemplary substitutions described herein. Thenon-cytotoxic and reduced cytotoxic variants of the cell-targetingmolecules of the present invention may be in certain situations moresuitable for delivery a heterologous CD8+ T-cell epitope and/oradditional exogenous materials than more cytotoxic variants.

The power of the immune system may be harnessed for therapeutic benefitby interventions which induce infection-like immune reactionsspecifically toward malignant cells (e.g. tumor cells) and/or malignanttissue loci (e.g. tumors) within a patient specifically such as, e.g.,by using a highly immunogenic, foreign epitope from infectious agent inorder to locally activate a variety of beneficial immune responses andto specifically mark targeted cells (e.g. tumor cells) as being foreignby inducing an imitation of an infected state. Alternatively, thisapproach could use highly immunogenic neoepitopes (derived from eitherinfectious or non-infectious agents) or highly immunogenic, non-selfepitopes derived from non-infectious agents, such as, e.g.,tumor-specific antigens, tumor-associated antigens, and molecules fromplants, fungi, etc. Furthermore, it may be possible to dictate whichcells or tissues the immune system is stimulated to by the choice ofepitope-peptide cargo(s). For example, using endogenous, non-self, tumorantigens (see e.g. Boon T, van der Bruggen P, J Exp Med 183: 725-9(1996); Vonderheide R et al., Immunity 10: 673-9 (1999); Van Der BruggenP et al., Immunol Rev 188: 51-64 (2002); Schreurs M et al., CancerImmunol Immunother 54: 703-12 (2005); Adotévi O et al., Clin Cancer Res12: 3158-67 (2006); Valentino M et al., J Immunol Methods 373: 111-26(2011)) to mark targeted cells may induce immune responses to untargetedcells displaying the same or related tumor-epitope whereas using viralepitopes to mark targeted cells in an uninfected cancer patient maylimit immune responses to only those cells which have had the epitopedelivered and presented in sufficient quantities and for sufficientdurations. In addition, it may possible to dictate what type of immuneresponse is induced by the choice of epitope-peptide cargo(s). Forexample, using endogenous, non-self, tumor antigens to mark targetedcells may induce anti-cancer immune responses to untargeted cellsdisplaying the same or related tumor-epitope whereas using viralepitopes to mark targeted cells in an uninfected cancer patient maylimit anti-viral type immune responses to only those cells which havehad the epitope delivered and presented in sufficient quantities and forsufficient durations.

The present invention provides immunotherapy methods involvingdelivering a CD8+ T-cell epitope-peptide cargo to a target cell in achordate and causing an immune response, the method comprising the stepof administering to the chordate a cell-targeting molecule orpharmaceutical composition of the present invention. For certain furtherembodiments, the immune response is an intercellular immune cellresponse selected from the group consisting of: CD8+ immune cellsecretion of a cytokine(s). CTL induced growth arrest in the targetcell, CTL induced necrosis of the target cell, CTL induced apoptosis ofthe target cell, non-specific cell death in a tissue locus,intermolecular epitope spreading, breaking immunological tolerance to amalignant cell type, and the chordate acquiring persistent immunity to amalignant cell-type (see e.g. Matsushita H et al., Cancer Immunol Res 3:26-36 (2015)). These immune responses can be detected and/or quantifiedusing techniques known to the skilled worker. For example, CD8+ immunecells can release immuno-stimulatory cytokines, such as, e.g., IFN-γ,tumor necrosis factor alpha (TNFα), macrophage inflammatory protein-1beta (MIP-1β), and interleukins such as IL-17, IL-4, IL-22, and IL-2(see e.g. Examples, infra; Seder R et al., Nat Rev Immunol 8: 247-58(2008)). IFN-γ can increase MHC class I molecule expression andsensitize neoplastic cells to CTL-mediated cell killing (Vlková V etal., Oncotarget 5: 6923-35 (2014)). Inflammatory cytokines can stimulatebystander T-cells that harbor unrelated TCR specificities to thecytokine releasing cell (see e.g. Tough D et al., Science 272: 1947-50(1996)). Activated CTLs can indiscriminately kill cells proximal toepitope-MHC class I complex presenting cell regardless of the proximalcell's present peptide-MHC class I complex repertoire (Wiedemann A etal., Proc Natl Acad Sci USA 103: 10985-90 (2006)). Thus, for certainfurther embodiments, the immune response is an intercellular immune cellresponse selected from the group consisting of: proximal cell killingmediated by immune cells where the proximal cell is not displaying anyCD8+ T-cell epitope-peptide delivered by the cell-targeting molecule ofthe present invention and regardless of the presence of anyextracellular target biomolecule of the binding region of thecell-targeting molecule physically coupled to the proximal cell(s) thatis killed.

The presence of non-self epitopes in CTL-lysed cells, whether targetcells or cells merely proximal to target cells, can be recognized andtargeted as foreign by the immune system, including recognition ofnon-self epitopes in target cells via the mechanism of intermolecularepitope spreading (see McCluskey J et al., Immunol Rev 164; 209-29(1998); Vanderlugt C et al., Immunol Rev 164: 63-72 (1998); VanderlugtC, Miller S, Nat Rev Immunol 2: 85-95 (2002)). Proximal cells mayinclude non-neoplastic cells, such as, e.g., cancer associatedfibroblasts, mesenchymal stem cells, tumor-associated endothelial cells,and immature myeloid-derived suppressor cells. For example, a cancercell may harbor on average 25 to 500 nonsynonymous mutations in codingsequences (see e.g. Fritsch E et al., Cancer Immunol Res 2: 522-9(2014)). Both cancer driver and non-driver mutations are part of themutational landscape of a cancer cell that corresponds to numerousnon-self epitopes per cell and the average tumor may possess ten or morenon-self epitopes (see e.g. Segal N et al., Cancer Res 68: 889-92(2008)). For example, mutant forms of the tumor protein p53 can containnon-self epitopes (see e.g. Vigneron N et al., Cancer Immun 13: 15(2013)). In addition, the presence of non-self epitopes, such as mutatedself-proteins, can result in the production of memory cells specific tothose new epitope(s). Because certain embodiments of the cell-targetingmolecules of the present invention may increase dendritic cell samplingat a targeted tissue locus, the probability of cross-priming the immunesystem with intracellular antigens may be increased (see e.g. Chiang Cet al., Erpert Opin Biol Ther 15: 569-82 (2015)). Thus, as a result ofcell-targeting molecule delivery of a heterologous, CD8+ T-cell epitopecargo and MHC class I presentation of that epitope, target cells andother proximal cells containing non-self epitopes can be rejected by theimmune system, including via non-self epitopes other than epitopesdelivered by a cell-targeting molecule of the invention. Such mechanismscould, e.g., induce antitumor immunity against tumor cells which do notexpress the extracellular target biomolecule of the binding region ofthe cell-targeting molecule.

Immune responses which involve cytokine secretion and/or T-cellactivation may result in modulation of the immuno-microenvironment of alocus within a chordate. A method of the present invention may be usedto alter the microenvironment of a tissue locus within a chordate inorder to change the regulatory homeostasis on immune cells, such as,e.g. tumor-associated macrophages, T-cells. T helper cells, antigenpresenting cells, and natural killer cells.

For certain embodiments, a method of the present invention may be usedto enhance anti-tumor cell immunity in a chordate subject and/or tocreate a persistent anti-tumor immunity in a chordate, such as, e.g.,due to the development of memory T-cells and/or alterations to the tumormicroenvironment.

Certain embodiments of the cell-targeting molecules of the presentinvention, or pharmaceutical compositions thereof, can be used to “seed”a locus within a chordate with non-self, CD8+ T-cell epitope-peptidepresenting cells in order to stimulate the immune system to police thelocus with greater strength and/or to alleviate immuno-inhibitorysignals, e.g., anergy inducing signals. In certain further embodimentsof this “seeding” method of the present invention, the locus is a tumormass or infected tissue site. In certain embodiments of this “seeding”method of the present invention, the non-self, CD8+ T-cellepitope-peptide is selected from the group consisting of: peptides notalready presented by the target cells of the cell-targeting molecule,peptides not present within any protein expressed by the target cell,peptides not present within the proteome or transcriptome of the targetcell, peptides not present in the extracellular microenvironment of thesite to be seeded, and peptides not present in the tumor mass or infecttissue site to be targeting.

This “seeding” method functions to label one or more target cells withina chordate with one or more MHC class I presented CD8+ T-cell epitopes(pMHC Is) for intercellular recognition by immune cells and activationof downstream immune responses. By exploiting the cell-internalizing,intracellularly routing, and/or MHC class I epitope delivering functionsof the cell-targeting molecules of the present invention, the targetcells that display the delivered CD8+ T-cell epitope can be recognizedby immunosurveillance mechanisms of the chordate's immune cells andresult in intercellular engagement of the presenting target cell by CD8+T-cells, such as, e.g., CTLs. This “seeding” method of using acell-targeting molecule of the present invention may stimulate immunecell mediated killing of target cells regardless of whether they arepresenting a cell-targeting molecule-delivered T-cell epitope(s), suchas, e.g., as a result of intermolecular epitope spreading and/orbreaking of immuno-tolerance to the target cell based on presentation ofendogenous antigens as opposed to artificially delivered epitopes. This“seeding” method of using a cell-targeting molecule of the presentinvention may provide a vaccination effect (new epitope(s) exposure)and/or vaccination-booster-dose effect (epitope re-exposure) by inducingadaptive immune responses to cells within the seeded microenvironment,such as, e.g. a tumor mass or infected tissue site, based on thedetection of epitopes which are either recognized as foreign by naïveT-cells and/or already recognizable as non-self (i.e. recall antigens)by memory T-cells. This “seeding” method may also induce the breaking ofimmuno-tolerance to a target cell population, a tumor mass, diseasedtissue site, and/or infected tissue site within a chordate, eitherperipherally or systemically.

The presence of dying or necrotic tumor cells at site or loci within achordate may result in a localized immune stimulation effect. Forexample, dying or necrotic tumor cells can release factors, such as,e.g., high mobility group proteins and/or ATP, which in turn canstimulate immunogenic maturation of immune cells. The seeding of a tumorlocus may also induce or increase the ectopic expression of ER proteins(e.g., calreticulin) on the plasma membrane of tumor cells which in turncan promote/increase MHC class antigen presentation and phagocytosis oftumor cells at that site.

Certain methods of the present invention involving the seeding of alocus within a chordate with one or more antigenic and/or immunogenicCD8+ T-cell epitopes may be combined with the administration ofimmunologic adjuvants, whether administered locally or systemically, tostimulate the immune response to certain antigens, such as, e.g., theco-administration of a composition of the present invention with one ormore immunologic adjuvants like a cytokine, bacterial product, or plantsaponin. Other examples of immunologic adjuvants which may be suitablefor use in the methods of the present invention include aluminum saltsand oils, such as, e.g., alums, aluminum hydroxide, mineral oils,squalene, paraffin oils, peanut oils, and thimerosal.

Certain methods of the present invention involve promoting immunogeniccross-presentation and/or cross-priming of naïve CD8+ T-cells in achordate. For certain methods of the present invention, cross-primingoccurs as a result of the death, and/or the manner of death, of a targetcell caused by a cell-targeting molecule of the present invention suchthat the exposure of intracellular antigens in the dying or dead targetcell to immunosurveillance mechanisms is promoted.

Because multiple, heterologous, CD8+ T-cell epitopes (either as cargosor as embedded or inserted regions of a Shiga toxin effector polypeptidecomponent) may be delivered by a single cell-targeting molecule of thepresent invention, a single embodiment of the cell-targeting molecule ofthe present invention may be therapeutically effective in differentindividual chordates of the same species with different MHC I classmolecule variants, such as, e.g., in humans with different HLA alleles.This ability of certain embodiments of the present invention may allowfor the combining within a single cell-targeting molecule of differentCD8+ T-cell epitope-peptides with different therapeutic effectiveness indifferent sub-populations of subjects based on MHC class I moleculediversity and polymorphisms. For example, human MHC class I molecules,the HLA proteins, vary among humans based on genetic ancestry, e.g.African (sub-Saharan), Amerindian, Caucasoid, Mongoloid. New Guinean andAustralian, or Pacific islander.

Cell-targeting molecules of the present invention which can deliverheterologous, CD8+ T-cell epitopes from CMV antigens may be particularlyeffective because a majority of the human population has specific setsof CD8+ T-cells primed to react to CMV antigens and are constantlyrepressing chronic CMV infections to remain asymptomatic for theirentire life. In addition, elderly humans may reactive even more quicklyand strongly to CMV CD8+ T-cell epitopes due to age-related changes inthe adaptive immune system regarding CMV, such as, e.g., a potentiallymore focused immune surveillance toward CMV and as shown by thecomposition of the T-cell antigen receptor repertoire and relative CTLlevels in more elderly humans (see e.g. Koch S et al., Ann NY Acad Sci1114: 23-35 (2007); Vescovini R et al., J Immunol 184: 3242-9 (2010):Cicin-Sain L et al., J Immunol 187: 1722-32 (2011); Fülöp T et al.,Front Immunol 4: 271 (2013); Pawelec G, Exp Gerontol 54: 1-5 (2014)).

C. Selective Cytotoxicity Among Cell-Types

Certain cell-targeting molecules of the present invention have uses inthe selective killing of specific target cells in the presence ofuntargeted, bystander cells. By targeting the delivery of immunogenic,CD8+ T-cell epitope cargos to the MHC class I pathway of target cells,the subsequent presentation of delivered CD8+ T-cell epitopes and theTCR specific regulation of CTL-mediated cytolysis of epitope-presentingtarget cells can be restricted to preferentially killing selectedcell-types in the presence of untargeted cells. In addition, the killingof target cells by the potent cytotoxic activity of various Shiga toxineffector polypeptides can be restricted to preferentially killing targetcells with the simultaneous delivery of an immunogenic T-cell epitopecargo and a cytotoxic toxin effector polypeptide.

For certain embodiments, upon administration of the cell-targetingmolecule of the present invention to a mixture of cell-types, thecell-targeting molecule is capable of selectively killing those cellswhich are physically coupled with an extracellular target biomoleculecompared to cell-types not physically coupled with an extracellulartarget biomolecule.

For certain embodiments, upon administration of the cell-targetingmolecule of the present invention to a mixture of cell-types, thecytotoxic cell-targeting molecule is capable of selectively killingthose cells which are physically coupled with an extracellular targetbiomolecule compared to cell-types not physically coupled with anextracellular target biomolecule. For certain embodiments, the cytotoxiccell-targeting molecule of the present invention is capable ofselectively or preferentially causing the death of a specific cell-typewithin a mixture of two or more different cell-types. This enablestargeting cytotoxic activity to specific cell-types with a highpreferentiality, such as a 3-fold cytotoxic effect, over “bystander”cell-types that do not express the target biomolecule. Alternatively,the expression of the target biomolecule of the binding region may benon-exclusive to one cell-type if the target biomolecule is expressed inlow enough amounts and/or physically coupled in low amounts withcell-types that are not to be targeted. This enables the targetedcell-killing of specific cell-types with a high preferentiality, such asa 3-fold cytotoxic effect, over “bystander” cell-types that do notexpress significant amounts of the target biomolecule or are notphysically coupled to significant amounts of the target biomolecule.

For certain further embodiments, upon administration of the cytotoxiccell-targeting molecule to two different populations of cell-types, thecytotoxic cell-targeting molecule is capable of causing cell death asdefined by the half-maximal cytotoxic concentration (CD₅₀) on apopulation of target cells, whose members express an extracellulartarget biomolecule of the binding region of the cytotoxic cell-targetingmolecule, at a dose at least three-times lower than the CD₅₀ dose of thesame cytotoxic cell-targeting molecule to a population of cells whosemembers do not express an extracellular target biomolecule of thebinding region of the cytotoxic cell-targeting molecule.

For certain embodiments, the cytotoxic activity of a cell-targetingmolecule of the present invention toward populations of cell-typesphysically coupled with an extracellular target biomolecule is at least3-fold higher than the cytotoxic activity toward populations ofcell-types not physically coupled with any extracellular targetbiomolecule of the binding region. According to the present invention,selective cytotoxicity may be quantified in terms of the ratio (alb) of(a) cytotoxicity towards a population of cells of a specific cell-typephysically coupled with a target biomolecule of the binding region to(b) cytotoxicity towards a population of cells of a cell-type notphysically coupled with a target biomolecule of the binding region.

For certain embodiments, the cytotoxicity ratio is indicative ofselective cytotoxicity which is at least 3-fold, 5-fold, 10-fold,15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 75-fold, 100-fold,250-fold, 500-fold, 750-fold, or I X)0-fold higher for populations ofcells or cell-types physically coupled with a target biomolecule of thebinding region compared to populations of cells or cell-types notphysically coupled with a target biomolecule of the binding region.

For certain embodiments, the preferential cell-killing function orselective cytotoxicity of a cell-targeting molecule of the presentinvention is due to an additional exogenous material (e.g. a cytotoxicmaterial) and/or heterologous, CD8+ T-cell epitope present in thecell-targeting molecule of the present invention and not necessarily aresult of the catalytic activity of a Shiga toxin effector polypeptidecomponent of the cell-targeting molecule.

It is important to note that for certain embodiments of thecell-targeting molecules of the present invention, upon administrationof the cell-targeting molecule to a chordate, the cell-targetingmolecule may cause the death of untargeted cells which are in thevicinity of a target cell and/or which are related to a target cell bysharing a common malignant condition. The presentation of certain T-cellepitopes by target cells within a chordate may result in CTL-mediatedkilling of the target cells as well as the killing of other cells notpresenting the delivered epitope but in the vicinity ofepitope-presenting cells. Additionally, the presentation of certainT-cell epitopes by targeted tumor cells within a chordate may result inintermolecular epitope spreading, re-programming of the tumormicroenvironment to stimulatory conditions, release of existing immunecells from anergy or removal of de-sensitization to target cells ordamaged tissues comprising them, and overcoming the physiological stateof tolerance of the subject's immune system to non-self tumor antigens(see Section X. Methods for Using a Cell-Targeting Molecule etc.,infra).

Administration of a cell-targeting molecule of the present invention toa chordate may be used in vivo to “clear” specific cell-types, such as,e.g., via immune system action/stimulation as in the form of targetingspecific-epitope restricted CTLs to kill targeted cell types and/orother cell types presenting the epitope, whether the epitope wasdelivered by the cell-targeting molecule or was already present beforeadministration.

D. Delivery of Additional Exogenous Material into the Interior of aTarget Cell

In addition to direct cell killing, cell-targeting molecules of thepresent invention optionally may be used for delivery of additionalexogenous materials into the interiors of target cells. The delivery ofadditional exogenous materials may be used, e.g., for cytotoxic,cytostatic, immune system stimulation, immune cell targeting,information gathering, and/or diagnostic functions. Non-cytotoxicvariants of the cytotoxic, cell-targeting molecules of the invention, oroptionally toxic variants, may be used to deliver additional exogenousmaterials to and/or label the interiors of cells physically coupled withan extracellular target biomolecule of the cell-targeting molecule.Various types of cells and/or cell populations which express targetbiomolecules to at least one cellular surface may be targeted by thecell-targeting molecules of the invention for receiving exogenousmaterials.

Because the cell-targeting molecules of the present invention, includingnontoxic forms thereof, are capable of entering cells physically coupledwith an extracellular target biomolecule recognized by its bindingregion, certain embodiments of the cell-targeting molecules of thepresent invention may be used to deliver additional exogenous materialsinto the interior of targeted cell-types. In one sense, the entirecell-targeting molecule of the invention is an exogenous material whichwill enter the cell; thus, the “additional” exogenous materials areheterologous materials linked to but other than the core cell-targetingmolecule itself. Non-toxic, cell-targeting molecules of the presentinvention which comprise a heterologous, CD8+ T-cell epitope-peptide(s)which does not stimulate CTL-mediated cell killing in certain situationsmay still be useful for delivering a “benign” CD8+T-cell-epitope-peptide which does not result in cell-killing upon MHCclass I presentation but allows for information gathering, such as,e.g., regarding immune system function in an individual MHC class Ivariant expression, and operability of the MHC class I system in acertain cell.

“Additional exogenous material” as used herein refers to one or moremolecules, often not generally present within a native target celland/or present at undesirably low levels, where the proteins of thepresent invention can be used to specifically transport such material tothe interior of a cell. Non-limiting examples of additional exogenousmaterials are cytotoxic agents, peptides, polypeptides, proteins,polynucleotides, detection promoting agents, and small moleculechemotherapeutic agents.

In certain embodiments of the proteins of the present invention fordelivery of additional exogenous material, the additional exogenousmaterial is a cytotoxic agent, such as, e.g., a small moleculechemotherapeutic agent, cytotoxic antibiotic, alkylating agent,antimetabolite, topoisomerase inhibitor, and/or tubulin inhibitor.Non-limiting examples of cytotoxic agents include aziridines,cisplatins, tetrazines, procarbazine, hexamethylmelamine, vincaalkaloids, taxanes, camptothecins, etoposide, doxorubicin, mitoxantrone,teniposide, novobiocin, aclarubicin, anthracyclines, actinomvcin,bleoimcin, plicamycin, mitomycin, daunorubicin, epirubicin, idarubicin,dolastatins, maytansines, docetaxel, adriamycin, calicheamicin,auristatins, pyrrolobenzodiazepine, carboplatin, 5-fluorouracil (5-FU),capecitabine, mitomycin C, paclitaxel,1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU), rifampicin, cisplatin,methotrexate, and gemcitabine.

In certain embodiments, the additional exogenous material comprises aprotein or polypeptide comprising an enzyme. In certain otherembodiments, the additional exogenous material is a nucleic acid, suchas, e.g a ribonucleic acid that functions as a small inhibiting RNA(siRNA) or microRNA (miRNA). In certain embodiments, the additionalexogenous material is an antigen, such as antigens derived frombacterial proteins, viral proteins, proteins mutated in cancer, proteinsaberrantly expressed in cancer, or T-cell complementary determiningregions. For example, exogenous materials include antigens, such asthose characteristic of antigen-presenting cells infected by bacteria,and T-cell complementary determining regions capable of functioning asexogenous antigens. Additional examples of exogenous materials includepolypeptides and proteins larger than an antigenic peptide, such asenzymes.

In certain embodiments, the additional exogenous material comprises aproapoptotic peptide, polypeptide, or protein, such as, e.g., BCL-2,caspases (e.g. fragments of caspase-3 or caspase-6), cytochromes,granzyme B, apoptosis-inducing factor (AIF), BAX, tBid (truncated Bid),and proapoptotic fragments or derivatives thereof (see e.g., Ellerby Het al., Nat Med 5: 1032-8 (1999); Mai J et al., Cancer Res 61: 7709-12(2001); Jia L et al., Cancer Res 63: 3257-62 (2003): Liu Y et al., MolCancer Ther 2: 1341-50 (2003): Perea S et al., Cancer Res 64: 7127-9(2004); Xu Y et al., J Immunol 173: 61-7 (2004); Dalken B et al., CellDeath Differ 13: 576-85 (2006); Wang T et al., Cancer Res 67: 11830-9(2007): Kwon M et al., Mol Cancer Ther 7: 1514-22 (2008); Shan L et al.,Cancer Biol Ther 11: 1717-22 (2008); Qiu X et al., Mol Cancer Ther 7:1890-9 (2008); Wang F et al., Clin Cancer Res 16: 2284-94 (2010); Kim Jet al., J Virol 85: 1507-16 (2011)).

Certain reduced-activity Shiga toxin effector polypeptides may beparticularly useful for delivering an additional exogenous material tocertain intracellular locations or subcellular compartments of targetcells.

E. Information Gathering for Diagnostic Functions

The cell-targeting molecules of the present invention may be used forinformation gathering functions. Certain embodiments of thecell-targeting molecules of the present invention may be used forimaging of specific pMHC I presenting cells using antibodies specific topMHC Is that recognize a heterologous, CD8+ T-cell epitope-peptide(delivered by a cell-targeting molecule of the present invention)complexed with a MHC class I molecule on a cell surface. In addition,certain cell-targeting molecules of the present invention have uses inthe in vitro and/or in vivo detection of specific cells, cell-types,and/or cell populations. In certain embodiments, the cell-targetingmolecules described herein are used for both diagnosis and treatment, orfor diagnosis alone.

The ability to conjugate detection promoting agents known in the art tovarious cell-targeting molecules of the present invention providesuseful compositions for the detection of cancer, tumor, growthabnormality, immune, and infected cells. These diagnostic embodiments ofthe cell-targeting molecules of the invention may be used forinformation gathering via various imaging techniques and assays known inthe art. For example, diagnostic embodiments of the cell-targetingmolecules of the invention may be used for information gathering viaimaging of intracellular organelles (e.g. endocytotic, Golgi,endoplasmic reticulum, and cytosolic compartments) of individual cancercells, immune cells, or infected cells in a patient or biopsy sample.

Various types of information may be gathered using the diagnosticembodiments of the cell-targeting molecules of the invention whether fordiagnostic uses or other uses. This information may be useful, forexample, in diagnosing neoplastic cell subtypes, determining MHC class Ipathway and/or TAP system functionality in specific cell-types,determining changes to MHC class I pathway and/or TAP systemfunctionality in specific cell-types over time, determining therapeuticsusceptibilities of a patient's disease, assaying the progression ofantineoplastic therapies over time, assaying the progression ofimmuno-modulatory therapies over time, assaying the progression ofantimicrobial therapies over time, evaluating the presence of infectedcells in transplantation materials, evaluating the presence of unwantedcell-types in transplantation materials, and/or evaluating the presenceof residual tumor cells after surgical excision of a tumor mass.

For example, subpopulations of patients might be ascertained usinginformation gathered using the diagnostic variants of the cell-targetingmolecules of the invention, and then individual patients could becategorized into subpopulations based on their unique characteristic(s)revealed using those diagnostic embodiments. For example, theeffectiveness of specific pharmaceuticals or therapies might be one typeof criterion used to define a patient subpopulation. For example, anontoxic diagnostic variant of a particular cytotoxic, cell-targetingmolecule of the invention may be used to differentiate which patientsare in a class or subpopulation of patients predicted to respondpositively to a cytotoxic variant of the same cell-targeting molecule ofthe invention. Accordingly, associated methods for patientidentification, patient stratification, and diagnosis usingcell-targeting molecules of the present invention, including non-toxicvariants of cytotoxic, cell-targeting molecules of the presentinvention, are considered to be within the scope of the presentinvention.

IV. Variations in the Polypeptide Sequence of the Protein Components ofthe Cell-Targeting Molecules of the Present Invention

The skilled worker will recognize that variations may be made to thecell-targeting molecules of the present invention described above, andpolynucleotides encoding any of the former, without diminishing theirbiological activities, e.g., by maintaining the overall structure andfunction of the cell-targeting molecules in delivering theirheterologous, CD8+ T-cell epitope-peptide cargos to the MHC class Ipresentation pathways of target cells after exogenous administration tothe target cells. For example, some modifications may facilitateexpression, facilitate purification, improve pharmacokinetic properties,and/or improve immunogenicity. Such modifications are well known to theskilled worker and include, for example, a methionine added at the aminoterminus to provide an initiation site, additional amino acids placed oneither terminus to create conveniently located restriction sites ortermination codons, and biochemical affinity tags fused to eitherterminus to provide for convenient detection and/or purification. Acommon modification to improve the immunogenicity of a polypeptide is toremove, after the production of the polypeptide, the starting methionineresidue, which may be formylated during production in a bacterial hostsystem, because, e.g., the presence of N-formylmethionine (fMet) mightinduce undesirable immune responses in chordates.

In certain variations of embodiments of the cell-targeting molecules ofthe invention, certain cell-targeting functionality of the bindingregion must be maintained so that the specificity and selectivity oftarget biomolecule binding is significantly preserved. In certainvariations of embodiments of the cell-targeting molecules of theinvention, certain biological activities of the Shiga toxin effectorpolypeptide may need to be preserved, e.g., inducing cellularinternalization, intracellular routing to certain subcellularcompartments (like compartments competent for entry into the MHC class Ipathway), and/or ability to deliver exogenous material(s) to certainsubcellular compartments of target cells.

Also contemplated herein is the inclusion of additional amino acidresidues at the amino and/or carboxy termini, such as sequences forbiochemical tags or other moieties. The additional amino acid residuesmay be used for various purposes including, e.g., to facilitate cloning,expression, post-translational modification, synthesis, purification,detection, and/or administration. Non-limiting examples of biochemicaltags and moieties are: chitin binding protein domains, enteropeptidasecleavage sites, Factor Xa cleavage sites, FIAsH tags, FLAG tags, greenfluorescent proteins (GFP), glutathione-S-transferase moieties, HA tags,maltose binding protein domains, myc tags, polyhistidine tags, ReAsHtags, strep-tags, strep-tag II, TEV protease sites, thioredoxin domains,thrombin cleavage site, and V5 epitope tags.

In certain of the above embodiments, the protein sequence of thecell-targeting molecules of the present invention, or polypeptidecomponents thereof, are varied by one or more conservative amino acidsubstitutions introduced into the protein or polypeptide component(s) aslong as the cell-targeting molecule retains the ability to deliver itsheterologous, CD8+ T-cell epitope-peptide cargo to a MHC class Ipresentation system of a target cell after exogenous administration tothe target cells such that the delivery and/or cell-surface MHC class Ipresentation of the delivered CD8+ T-cell epitope is detectable using anassay known to the skilled worker and/or described herein.

As used herein, the term “conservative substitution” denotes that one ormore amino acids are replaced by another, biologically similar aminoacid residue.

Examples include substitution of amino acid residues with similarcharacteristics, e.g small amino acids, acidic amino acids, polar aminoacids, basic amino acids, hydrophobic amino acids, and aromatic aminoacids (see, for example, Table B, infra). An example of a conservativesubstitution with a residue normally not found in endogenous, mammalianpeptides and proteins is the conservative substitution of an arginine orlysine residue with, for example, omithine, canavanine,aminoethvlcysteine, or another basic amino acid. For further informationconcerning phenotypically silent substitutions in peptides and proteinssee, e.g., Bowie J et al., Science 247: 1306-10 (1990).

TABLE B Examples of Conservative Amino Acid  Substitutions I II III IV VVI VII VIII IX X XI XII XIII XIV A D H C F N A C F A C A A D G E K I W QG M H C D C C E P Q R L Y S I P W F E D D G S N M T L Y G H G E K T V VH  K N G P I N P H Q L Q S K R M R T N S R S V Q T T T R V S W P Y T

In the conservative substitution scheme in Table B above, exemplaryconservative substitutions of amino acids are grouped by physicochemicalproperties—I: neutral, hydrophilic: II: acids and amides; III: basic;IV: hydrophobic; V: aromatic, bulky amino acids, VI hydrophilicuncharged. VII aliphatic uncharged, VIII non-polar uncharged, IXcycloalkenyl-associated, X hydrophobic, XI polar, XII small, XIIIturn-permitting, and XIV flexible. For example, conservative amino acidsubstitutions include the following: 1) S may be substituted for C; 2) Mor L may be substituted for F; 3) Y may be substituted for M: 4) Q or Emay be substituted for K; 5) N or Q may be substituted for H: and 6) Hmay be substituted for N.

In certain embodiments, the cell-targeting molecules of the presentinvention (e.g. cell-targeting fusion proteins) may comprise functionalfragments or variants of a polypeptide region of the invention thathave, at most, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidresidue substitutions compared to a polypeptide sequence recited herein,as long as the cell-targeting molecule comprising it is capable ofdelivering its heterologous, CD8+ T-cell epitope-peptide cargo to a MHCclass I presentation pathway of a target cell. Variants of thecell-targeting molecules of the invention are within the scope of thepresent invention as a result of changing a polypeptide component of thecell-targeting protein of the invention by altering one or more aminoacids or deleting or inserting one or more amino acids, such as withinthe binding region or the Shiga toxin effector polypeptide component, inorder to achieve desired properties, such as changed cytotoxicity,changed cytostatic effects, changed immunogenicity, and/or changed serumhalf-life. A cell-targeting molecule of the invention, or polypeptidecomponent thereof, may further be with or without a signal sequence.

Accordingly, in certain embodiments, the binding region ofcell-targeting molecules of the present invention comprises or consistsessentially of amino acid sequences having at least 80%, 85%, 90%, 95%,97%, 98%, 99%, 99.5% or 99.7% overall sequence identity to a bindingregion recited herein or otherwise already known when compared to analigned sequence in which the alignment is done by a computer homologyprogram known in the art, as long as the binding region exhibits, as acomponent of the cell-targeting molecule, a reasonable amount ofextracellular target biomolecule binding specificity and affinity, suchas, e.g. by exhibiting a K_(D) to the target biomolecule of 10⁻⁵ to10⁻¹² moles/liter.

In certain embodiments, the Shiga toxin effector polypeptide region ofcell-targeting molecules of the present invention comprises or consistsessentially of amino acid sequences having at least 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5% or 99.7% overall sequenceidentity to a naturally occurring toxin, such as, e.g., Shiga toxin ASubunit, such as any one of SEQ ID NOs: 1-18, when compared to analigned sequence in which the alignment is done by a computer homologyprogram known in the art, as long as the Shiga toxin effectorpolypeptide exhibits, as a component of the cell-targeting molecule, therequired level of the Shiga toxin effector function(s) related tointracellular delivery of a the cell-targeting molecule's heterologous,CD8+ T-cell epitope-peptide cargo to the MHC class I presentationpathway of at least one target cell-type.

In certain embodiments, the Shiga toxin effector polypeptide componentsof the cell-targeting molecules of the present invention may be alteredto change the enzymatic activity and/or cytotoxicity of the Shiga toxineffector polypeptide, as long as the Shiga toxin effector polypeptideexhibits, as a component of the cell-targeting molecule, the requiredlevel of the Shiga toxin effector function(s) related to intracellulardelivery of a the cell-targeting molecule's CD8+ T-cell epitope-peptidecargo to the MHC class I presentation pathway of at least one targetcell-type. This change may or may not result in a change in thecytotoxicity of the Shiga toxin effector polypeptide or cell-targetingmolecule of which the altered Shiga toxin effector polypeptide is acomponent. Both Shiga toxin enzymatic activity and cytotoxicity may bealtered, reduced, or eliminated by mutation or truncation. Possiblealterations include mutations to the Shiga toxin effector polypeptideselected from the group consisting of: a truncation, deletion,inversion, insertion, rearrangement, and substitution as long as theShiga toxin effector polypeptide retains, as a component of thecell-targeting molecule, the required level of the Shiga toxin effectorfunction(s) related to intracellular delivery of a the cell-targetingmolecule's heterologous, CD8+ T-cell epitope-peptide cargo to the MHCclass I presentation pathway of at least one target cell-type.

The cytotoxicity of the A Subunits of members of the Shiga toxin familymay be altered, reduced, or eliminated by mutation or truncation. Thecell-targeting molecules of the present invention each comprise a Shigatoxin A Subunit effector polypeptide region which provide eachcell-targeting molecule the ability to deliver the cell-targetingmolecule's heterologous, CD8+ T-cell epitope-peptide cargo to the MHCclass I presentation pathway of at least one target cell-type regardlessof Shiga toxin effector polypeptide catalytic activity. As shown in theExamples below, the catalytic activity and cytotoxicity of Shiga toxineffector polypeptides may be uncoupled from other Shiga toxin effectorfunctions required to provide a cell-targeting molecule of the presentinvention with the ability to deliver a fused, heterologous, CD8+ T-cellepitope to the MHC class I presentation pathway of a target cell-type.Thus in certain embodiments of the cell-targeting molecules of thepresent invention, the Shiga toxin effector polypeptide component isengineered to exhibit diminished or abolished Shiga toxin cytotoxicity,such as, e.g., due to the presence of amino acid residue mutationsrelative to a wild-type Shiga toxin A Subunit in one or more keyresidues involved in enzymatic activity. This provides cell-targetingmolecules of the invention which do not kill target cells directly viathe Shiga toxin function of cytotoxicity. Such cell-targeting moleculesof the invention, which lack cytotoxic Shiga toxin effector polypeptideregions, are useful for effectuating 1) cell-killing via the delivery ofa heterologous, CD8+ T-cell epitope-peptide for MHC class I presentationby a target cell, 2) the stimulation of desirable, intercellular immunecell response(s) to a target cells as a result of the delivery of aheterologous, CD8+ T-cell epitope-peptide to the MHC class I system oftarget cells, and/or 3) the labeling of target cells with specific CD8+T-cell epitope-peptide/MHC class I molecule complexes when the targetcell is not defective in the machinery required to do so.

The catalytic and/or cytotoxic activity of the A Subunits of members ofthe Shiga toxin family may be diminished or eliminated by mutation ortruncation. The most critical residues for enzymatic activity and/orcytotoxicity in the Shiga toxin A Subunits have been mapped to thefollowing residue-positions: aspargine-75, tyrosine-77, glutamate-167,arginine-170, arginine-176, and tryptophan-203 among others (Di R etal., Toxicon 57: 525-39 (2011)). In particular, a double-mutantconstruct of Stx2A containing glutamate-E167-to-lysine andarginine-176-to-lysine mutations was completely inactivated; whereas,many single mutations in Stx1 and Stx2 showed a 10-fold reduction incytotoxicity. The positions labeled tyrosine-77, glutamate-167,arginine-170, tyrosine-114, and tryptophan-203 have been shown to beimportant for the catalytic activity of Stx, Stx1, and Stx2 (Hovde C etal., Proc Natl Acad Sci USA 85: 2568-72 (1988): Deresiewicz R et al.,Biochemistry 31: 50 3272-80 (1992); Deresiewicz R et al., Mol Gen Genet241: 467-73 (1993); Ohmura M et al., Microb Pathog 15: 169-76 (1993):Cao C et al., Microbiol Immunol 38: 441-7 (1994); Suhan M, Hovde C.,Infect Immun 66: 5252-9 (1998)). Mutating both glutamate-167 andarginine-170 eliminated the enzymatic activity of Sit-I A1 in acell-free ribosome inactivation assay (LaPointe P et al., J Biol Chem280: 23310-18 (2005)). In another approach using de novo expression ofSlt-I A1 in the endoplasmic reticulum, mutating both glutamate-167 andarginine-170 eliminated Slt-I A1 fragment cytotoxicity at thatexpression level (LaPointe P et al., J Biol Chem 280: 23310-18 (2005)).

Further, truncation of Stx1A to 1-239 or 1-240 reduced its cytotoxicity,and similarly, truncation of Stx2A to a conserved hydrophobic residuereduced its cytotoxicity. The most critical residues for bindingeukaryotic ribosomes and/or eukaryotic ribosome inhibition in the Shigatoxin A Subunit have been mapped to the following residue-positionsarginine-172, arginine-176, arginine-179, arginine-188, tyrosine-189,valine-191, and leucine-233 among others (McCluskey A et al., PLoS One7: e31191 (2012)).

In certain embodiments of the cell-targeting molecules of the invention,the Shiga toxin A Subunit effector polypeptide derived from orcomprising a component derived from a Shiga toxin A Subunit (e.g. anyone of SEQ ID NOs: 1-18) comprises an alteration from a wild-type Shigatoxin, polypeptide sequence, such as, e.g., one or more of the followingamino acid residue substitution(s): asparagine at position 75, tyrosineat position 77, tyrosine at position 114, glutamate at position 167,arginine at position 170, arginine at position 176, tryptophan atposition 202, and/or substitution of the tryptophan at position 203.Examples of such substitutions will be known to the skilled worker basedon the prior art, such as asparagine at position 75 to alanine, tyrosineat position 77 to serine, substitution of the tyrosine at position 114to alanine, substitution of the glutamate at position 167 to aspartate,substitution of the arginine at position 170 to alanine, substitution ofthe arginine at position 176 to lysine, and/or substitution of thetryptophan at position 203 to alanine. Other mutations which eitherenhance or reduce Shiga toxin A Subunit effector polypeptide enzymaticactivity and/or cytotoxicity are within the scope of the presentinvention and may be determined using well known techniques and assaysdisclosed herein.

In certain embodiments, the cell-targeting molecule of the presentinvention, or a proteinaceous component thereof, comprises one or morepost-translational modifications, such as, e.g., phosphorylation,acetylation, glycosylation, amidation, hydroxylation, and/or methylation(see e.g. Nagata K et al., Bioinformatics 30: 1681-9 (2014)).

In certain embodiments of the cell-targeting molecules of the presentinvention, one or more amino acid residues may be mutated, inserted, ordeleted in order to increase the enzymatic activity of the Shiga toxineffector polypeptide region as long as the cell-targeting molecule iscapable of delivering its heterologous, CD8+ T-cell epitope-peptidecargo to the MHC class I presentation pathway of a target cell. Forexample, mutating residue-position alanine-231 in Stx1A to glutamateincreased its enzymatic activity in vitro (Suhan M. Hovde C. InfectImmun 66: 5252-9 (1998)).

The cell-targeting molecules of the present invention may optionally beconjugated to one or more additional agents, which may includetherapeutic and/or diagnostic agents known in the art, including suchagents as described herein.

V. Production, Manufacture, and Purification of Cell-Targeting Moleculesof the Present Invention

The cell-targeting molecules of the present invention may be producedusing biochemical engineering techniques well known to those of skill inthe art. For example, cell-targeting molecules of the invention and/orprotein components thereof may be manufactured by standard syntheticmethods, by use of recombinant expression systems, or by any othersuitable method. Thus, certain cell-targeting molecules of the presentinvention, and protein components thereof, may be synthesized in anumber of ways, including, e.g methods comprising: (1) synthesizing apolypeptide or polypeptide component of a protein using standardsolid-phase or liquid-phase methodology, either stepwise or by fragmentassembly, and isolating and purifying the final polypeptide or proteincompound product: (2) expressing a polynucleotide that encodes apolypeptide or polypeptide component of a cell-targeting molecule of theinvention in a host cell and recovering the expression product from thehost cell or host cell culture: or (3) cell-free in vitro expression ofa polynucleotide encoding a polypeptide or polypeptide component of acell-targeting molecule of the invention, and recovering the expressionproduct: or by any combination of the methods of (1), (2) or (3) toobtain fragments of the peptide component, subsequently joining (e.g.ligating) the fragments to obtain the peptide component, and recoveringthe peptide component. For example, polypeptide and/or peptidecomponents may be ligated together using coupling reagents, such as,e.g., N,N′-dicyclohexycarbodiimide andN-ethyl-5-phenyl-isoxazolium-3′-sulfonate (Woodward's reagent K).

It may be preferable to synthesize a cell-targeting molecule or aproteinaceous component of a cell-targeting molecule of the invention bymeans of solid-phase or liquid-phase peptide synthesis. Cell-targetingmolecules of the invention and components thereof may suitably bemanufactured by standard synthetic methods. Thus, peptides may besynthesized by, e.g. methods comprising synthesizing the peptide bystandard solid-phase or liquid-phase methodology, either stepwise or byfragment assembly, and isolating and purifying the final peptideproduct. In this context, reference may be made to WO1998/11125 or,inter alia, Fields G et al., Principles and Practice of Solid-PhasePeptide Synthesis (Synthetic Peptides, Grant G, ed., Oxford UniversityPress, U.K., 2nd ed., 2002) and the synthesis examples therein.

Cell-targeting molecules of the present invention which are fusionproteins may be prepared (produced and purified) using recombinanttechniques well known in the art. In general, methods for preparingproteins by culturing host cells transformed or transfected with avector comprising the encoding polynucleotide and recovering the proteinfrom cell culture are described in, e.g. Sambrook J et al., MolecularCloning: A Laboratory Man al (Cold Spring Harbor Laboratory Press, NY,U.S., 1989); Dieffenbach C et al., PCR Primer: A Laboratory Manual (ColdSpring Harbor Laboratory Press, N.Y., U.S., 1995). Any suitable hostcell may be used to produce a cell-targeting protein of the presentinvention or a proteinaceous component of a cell-targeting molecule ofthe present invention. Host cells may be cells stably or transientlytransfected, transformed, transduced or infected with one or moreexpression vectors which drive expression of a cell-targeting moleculeof the present invention and/or protein component thereof. In addition,a cell-targeting molecule of the present invention may be produced bymodifying the polynucleotide encoding the cell-targeting protein of thepresent invention or a proteinaceous component of a cell-targetingmolecule of the present invention that result in altering one or moreamino acids or deleting or inserting one or more amino acids in order toachieve desired properties, such as changed cytotoxicity, changedcytostatic effects, and/or changed serum half-life.

There are a wide variety of expression systems which may be chosen toproduce a cell-targeting molecule of the present invention. For example,host organisms for expression of cell-targeting proteins of theinvention include prokaryotes, such as E. coli and B. subtilis,eukaryotic cells, such as yeast and filamentous fungi (like S.cerevistae, P. pastoris, A. awamori, and K. lactis), algae (like C.reinhardtii), insect cell lines, mammalian cells (like CHO cells), plantcell lines, and eukaryotic organisms such as transgenic plants (like A.thaliana and N. benthamiana) (see e.g. Zarschler K et al., MicrobialCell Factories 12: 97 (2013)).

Accordingly, the present invention also provides methods for producing acell-targeting molecule of the present invention according to aboverecited methods and using (i) a polynucleotide encoding part or all of amolecule of the invention or a polypeptide component of a cell-targetingmolecule of the present invention, (ii) an expression vector comprisingat least one polynucleotide of the invention capable of encoding part orall of a molecule of the invention or a polypeptide component thereofwhen introduced into a suitable host cell or cell-free expressionsystem, and/or (iii) a host cell comprising a polynucleotide orexpression vector of the invention.

When a protein is expressed using recombinant techniques in a host cellor cell-free system, it is advantageous to separate (or purify) thedesired protein away from other components, such as host cell factors,in order to obtain preparations that are of high purity or aresubstantially homogeneous. Purification can be accomplished by methodswell known in the art, such as centrifugation techniques, extractiontechniques, chromatographic and fractionation techniques (e.g. sizeseparation by gel filtration, charge separation by ion-exchange column,hydrophobic interaction chromatography, reverse phase chromatography,chromatography on silica or cation-exchange resins such as DEAE and thelike, chromatofocusing, and Protein A Sepharose chromatography to removecontaminants), and precipitation techniques (e.g. ethanol precipitationor ammonium sulfate precipitation). Any number of biochemicalpurification techniques may be used to increase the purity of acell-targeting molecule of the present invention. In certainembodiments, the cell-targeting molecules of the invention mayoptionally be purified in homo-multimeric forms (e.g. a stable complexof two or more identical cell-targeting molecules of the invention) orin hetero-multimeric forms (e.g. a stable complex of two or morenon-identical cell-targeting molecules of the invention).

In the Examples below are descriptions of non-limiting examples ofmethods for producing a cell-targeting molecule of the present inventionor polypeptide component thereof, as well as specific but non-limitingaspects of production for exemplary cell-targeting molecules of thepresent invention.

VI. Pharmaceutical and Diagnostic Compositions Comprising aCell-Targeting Molecule of the Present Invention

The present invention provides cell-targeting molecules for use, aloneor in combination with one or more additional therapeutic agents, in apharmaceutical composition, for treatment or prophylaxis of conditions,diseases, disorders, or symptoms described in further detail below (e.g.cancers, malignant tumors, non-malignant tumors, growth abnormalities,immune disorders, and microbial infections). The present inventionfurther provides pharmaceutical compositions comprising a cell-targetingmolecule of the invention, or a pharmaceutically acceptable salt orsolvate thereof, according to the invention, together with at least onepharmaceutically acceptable carrier, excipient, or vehicle. In certainembodiments, the pharmaceutical composition of the present invention maycomprise homo-multimeric and/or hetero-multimeric forms of thecell-targeting molecules of the invention. The pharmaceuticalcompositions will be useful in methods of treating, ameliorating, orpreventing a disease, condition, disorder, or symptom described infurther detail below. Each such disease, condition, disorder, or symptomis envisioned to be a separate embodiment with respect to uses of apharmaceutical composition according to the invention. The inventionfurther provides pharmaceutical compositions for use in at least onemethod of treatment according to the invention, as described in moredetail below.

As used herein, the terms “patient” and “subject” are usedinterchangeably to refer to any organism, commonly vertebrates such ashumans and animals, which presents symptoms, signs, and/or indicationsof at least one disease, disorder, or condition. These terms includemammals such as the non-limiting examples of primates, livestock animals(e.g. cattle, horses, pigs, sheep, goats, etc.), companion animals (e.g.cats, dogs, etc.) and laboratory animals (e.g. mice, rabbits, rats,etc.).

As used herein, “treat,” “treating,” or “treatment” and grammaticalvariants thereof refer to an approach for obtaining beneficial ordesired clinical results. The terms may refer to slowing the onset orrate of development of a condition, disorder or disease, reducing oralleviating symptoms associated with it, generating a complete orpartial regression of the condition, or some combination of any of theabove. For the purposes of this invention, beneficial or desiredclinical results include, but are not limited to, reduction oralleviation of symptoms, diminishment of extent of disease,stabilization (e.g. not worsening) of state of disease, delay or slowingof disease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. “Treat,” “treating,” or “treatment” can also meanprolonging survival relative to expected survival time if not receivingtreatment. A subject (e.g. a human) in need of treatment may thus be asubject already afflicted with the disease or disorder in question. Theterms “treat.” “treating.” or “treatment” includes inhibition orreduction of an increase in severity of a pathological state or symptomsrelative to the absence of treatment, and is not necessarily meant toimply complete cessation of the relevant disease, disorder, orcondition. With regard to tumors and/or cancers, treatment includesreductions in overall tumor burden and/or individual tumor size.

As used herein, the terms “prevent,” “preventing,” “prevention” andgrammatical variants thereof refer to an approach for preventing thedevelopment of, or altering the pathology of a condition, disease, ordisorder. Accordingly, “prevention” may refer to prophylactic orpreventive measures. For the purposes of this invention, beneficial ordesired clinical results include, but are not limited to, prevention orslowing of symptoms, progression or development of a disease, whetherdetectable or undetectable. A subject (e.g. a human) in need ofprevention may thus be a subject not yet afflicted with the disease ordisorder in question. The term “prevention” includes slowing the onsetof disease relative to the absence of treatment, and is not necessarilymeant to imply permanent prevention of the relevant disease, disorder orcondition. Thus “preventing” or “prevention” of a condition may incertain contexts refer to reducing the risk of developing the condition,or preventing or delaying the development of symptoms associated withthe condition.

As used herein, an “effective amount” or “therapeutically effectiveamount” is an amount or dose of a composition (e.g. a therapeuticcomposition or agent) that produces at least one desired therapeuticeffect in a subject, such as preventing or treating a target conditionor beneficially alleviating a symptom associated with the condition. Themost desirable therapeutically effective amount is an amount that willproduce a desired efficacy of a particular treatment selected by one ofskill in the art for a given subject in need thereof. This amount willvary depending upon a variety of factors understood by the skilledworker, including but not limited to the characteristics of thetherapeutic compound (including activity, pharmacokinetics,pharmacodynamics, and bioavailability), the physiological condition ofthe subject (including age, sex, disease type, disease stage, generalphysical condition, responsiveness to a given dosage, and type ofmedication), the nature of the pharmaceutically acceptable carrier orcarriers in the formulation, and the route of administration. Oneskilled in the clinical and pharmacological arts will be able todetermine a therapeutically effective amount through routineexperimentation, namely by monitoring a subject's response toadministration of a compound and adjusting the dosage accordingly (seee.g. Remington: The Science and Practice of Pharmacy (Gennaro A, ed.,Mack Publishing Co., Easton, Pa., U.S., 19th ed., 1995)).

A pharmaceutical composition of the present invention optionallyincludes a pharmaceutically acceptable excipient. Non-limiting examplesof pharmaceutically acceptable excipients include arginine, argininesulfate, citric acid, glycerol, hydrochloric acid, mannitol, methionine,polysorbate, sodium chloride, sodium citrate, sodium hydroxide,sorbitol, sucrose, trehalose, and/or water. In certain embodiments, thepharmaceutical composition of the present invention comprises an aqueouscarrier and at least one pharmaceutically acceptable excipient. Incertain other embodiments, the pharmaceutical composition of the presentinvention comprises a salt and/or powder, such as, e.g. a freeze-dried,lyophilized, dehydrated, and/or cryodesiccated composition comprising atleast one pharmaceutically acceptable excipient. In certain embodimentsof the pharmaceutical composition of the present invention, theexcipient functions to reduce and/or limit the immunogenicity and/orimmunogenic potential of the cell-targeting molecule, such as, e.g.after administration and/or repeated administration to a mammal.

The pharmaceutical compositions of the present invention may compriseone or more adjuvants such as a buffer, tonicity-adjusting agent(isotonic agent), antioxidant, surfactant, stabilizer, preservative,emulsifying agent, cryoprotective agent, wetting agent, and/ordispersing agent or other additives well known to those of skill in theart, such as, e.g. a binding agent. In certain embodiments, thepharmaceutical composition of the present invention comprises an aqueouscarrier and a pharmaceutically acceptable adjuvant or other additive. Incertain other embodiments, the pharmaceutical composition of the presentinvention comprises a salt and/or powder, such as, e.g. a freeze-dried,lyophilized, dehydrated, and/or crvodesiccated composition comprising apharmaceutically acceptable adjuvant or other additive. Non-limitingexamples of pharmaceutically suitable stabilizers include human albuminand polysorbates such as, e.g., polyoxyethylene (20) sorbitanmonolaurate (polysorbate 20), polyoxyethylene (20) sorbitanmonopalmitate (polysorbate 40), polyoxyethylene (20) sorbitanmonostearate (polysorbate 60), and (polyoxyethylene (20) sorbitanmonooleate (polysorbate 80).

The pharmaceutical composition of the present invention may comprise oneor more pharmaceutically acceptable buffers. Non-limiting examples ofsuitable buffers include acetate, citrate, citric acid, histidine,phosphate, sodium citrate, and succinate buffers. In certainembodiments, the pharmaceutical composition of the present inventioncomprises an aqueous carrier comprising a pharmaceutically acceptablebuffer. In certain other embodiments, the pharmaceutical composition ofthe present invention comprises a salt and/or powder, such as, e.g. afreeze-dried, lyophilized, dehydrated, and/or cryodesiccated compositioncomprising a pharmaceutically acceptable buffer.

The pharmaceutical composition of the present invention may comprise oneor more pharmaceutically acceptable isotonic agents ortonicity-adjusting agents. Non-limiting examples of suitable isotonicagents include sugars (e.g. dextrose), sugar alcohols, sodium chloride,and the like. Further examples of suitable sugars include disaccharideslike sucrose and trehalose. Exemplary, pharmaceutically acceptable sugaralcohols include glycerol, mannitol, and sorbitol. In certainembodiments, the pharmaceutical composition of the present inventioncomprises an aqueous carrier and a pharmaceutically acceptable isotonicagent. In certain other embodiments, the pharmaceutical composition ofthe present invention comprises a salt and/or powder, such as, e.g. afreeze-dried, lyophilized, dehydrated, and/or cryodesiccated compositioncomprising a pharmaceutically acceptable isotonic agent.

The pharmaceutical compositions of the present invention may compriseone or more pharmaceutically acceptable antioxidants. Exemplarypharmaceutically acceptable antioxidants include water solubleantioxidants, such as, e.g., ascorbic acid, cysteine hydrochloride,methionine, sodium bisulfate, sodium metabisulfite, sodium sulfite andthe like; oil-soluble antioxidants, such as, e.g., ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propylgallate, alpha-tocopherol, and the like; andmetal-chelating agents, such as, e.g., citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, andthe like. In certain embodiments, the pharmaceutical composition of thepresent invention comprises an aqueous carrier and a pharmaceuticallyacceptable antioxidant. In certain other embodiments, the pharmaceuticalcomposition of the present invention comprises a salt and/or powder,such as, e.g. a freeze-dried, lyophilized, dehydrated, and/orcryodesiccated composition comprising a pharmaceutically acceptableantioxidant.

A pharmaceutical composition of the present invention may comprise oneor more pharmaceutically acceptable surfactants and/or emulsifyingagents (emulsifiers). Non-limiting examples of suitable surfactantsand/or emulsifiers include polysorbates such as, e.g., polyoxyethylene(20) sorbitan monolaurate (polysorbate 20), polyoxyethylene (20)sorbitan monopalmitate (polysorbate 40), polyoxyethylene (20) sorbitanmonostearate (polysorbate 60), and (polyoxyethylene (20) sorbitanmonooleate (polysorbate 80). In certain embodiments, the pharmaceuticalcomposition of the present invention comprises an aqueous carrier and apharmaceutically acceptable surfactant and/or emulsifier. In certainother embodiments, the pharmaceutical composition of the presentinvention comprises a salt and/or powder, such as, e.g. a freeze-dried,lyophilized, dehydrated, and/or cryodesiccated composition comprising apharmaceutically acceptable surfactant and/or emulsifier. One or moresurfactants and/or emulsifying agents may also be desirable in apharmaceutical composition of the present invention to help preventaggregation of the cell-targeting molecule of the present invention. Thepharmaceutical compositions of the present invention may comprise one ormore pharmaceutically acceptable preservative agents. For example,preventing the presence of microorganisms may be ensured both bysterilization procedures, and by the inclusion of various antibacterialand antifungal agents, such as, e.g., paraben, chlorobutanol, phenolsorbic acid, and the like in the compositions of the present invention.

A pharmaceutical composition of the present invention may comprise oneor more pharmaceutically acceptable cryoprotective agents, also referredto as cryoprotectants or cryogenic protectants. Non-limiting examples ofsuitable cryoprotectants include ethylene glycol, glycerol, sorbitol,sucrose, and trehalose. In certain embodiments, the pharmaceuticalcomposition of the present invention comprises an aqueous carrier and apharmaceutically acceptable cryoprotectant. In certain otherembodiments, the pharmaceutical composition of the present inventioncomprises a salt and/or powder, such as, e.g. a freeze-dried,lyophilized, dehydrated, and/or cryodesiccated composition comprising apharmaceutically acceptable cryoprotectant.

In addition, prolonged absorption of the injectable pharmaceutical formmay be brought about by the inclusion of agents which delay absorptionsuch as, e.g., a monostearate salt, aluminum monostearate, and/orgelatin.

In another aspect, the present invention provides pharmaceuticalcompositions comprising one or a combination of different polypeptidesand/or cell-targeting molecules of the invention, or an ester, salt oramide of any of the foregoing, and at least one pharmaceuticallyacceptable carrier.

The pH of the pharmaceutical composition of the present invention can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide, or buffers with acetate, citrate, citric acid, histidine,sodium citrate, succinate, phosphate, and the like. Non-limitingexamples of pharmaceutically acceptable solvents or carriers for use ina pharmaceutical composition of the present invention include aqueoussolutions comprising a cell-targeting molecule of the present inventionand a buffer such as, e.g., citrate, histidine, phosphate, or succinateadjusted to pH 5.0, 6.0, 7.0, or 4.0, respectively. Certain embodimentsof the present invention include compositions comprising one of theaforementioned solvents and/or carriers of the present invention.

Pharmaceutical compositions of the present invention that are solutionsor suspensions used for intradermal or subcutaneous applicationtypically include one or more of: a sterile diluent such as water forinjection, saline solution, fixed oils, polyethylene glycols, glycerine,propylene glycol or other synthetic solvents: antibacterial agents suchas benzyl alcohol or methyl parabens; antioxidants such as ascorbicacid, cysteine hydrochloride, methionine, sodium bisulfate, sodiummetabisulfite, and sodium sulfite: chelating agents such as citric acid,ethylenediaminetetraacetic acid, sorbitol, tartaric acid, and phosphoricacid: surfactants such as a polysorbate; buffers such as acetate,citrate, histidine, and phosphate buffers; and tonicity adjusting agentssuch as, e.g., dextrose, glycerol, mannitol, sodium chloride, sorbitol,sucrose, and trehalose. Such preparations may be enclosed in ampoules,disposable syringes or multiple dose vials made of a glass or plastic.

Sterile injectable solutions may be prepared by incorporating a proteinor cell-targeting molecule of the present invention in the requiredamount in an appropriate solvent with one or a combination ofingredients described above, as required, followed by sterilizationmicrofiltration. Dispersions may be prepared by incorporating the activecompound into a sterile vehicle that contains a dispersion medium andother ingredients, such as those described above. In the case of sterilepowders for the preparation of sterile injectable solutions, the methodsof preparation are vacuum drying and freeze-drying (lyophilization) thatyield a powder of the active ingredient in addition to any additionaldesired ingredient from a sterile-filtered solution thereof. In certainembodiments, the pharmaceutical composition of the present inventioncomprises a powder comprising sorbitol, trehalose, sodium citrate, andpolysorbate-20, and optionally, further comprises glycerol and/ormethionine. In certain embodiments, the pharmaceutical composition ofthe present invention comprises sodium citrate, trehalose, andpolysorbate-20, and optionally, further comprises glycerol and/ormethionine.

In certain embodiments, the pharmaceutical composition of the presentinvention comprises sorbitol, sodium citrate, and polysorbate-20, andoptionally, further comprises albumin, glycerol, and/or methionine. Incertain embodiments, the pharmaceutical composition of the presentinvention comprises sorbitol, histidine, and polysorbate-20, andoptionally, further comprises albumin, glycerol, and/or methionine.

The formulations of the pharmaceutical compositions of the presentinvention may conveniently be presented in unit dosage form and may beprepared by any of the methods well known in the art of pharmacy. Insuch form, the composition is divided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofthe preparations, for example, packeted tablets, capsules, and powdersin vials or ampoules. The unit dosage form can also be a capsule,cachet, or tablet itself, or it can be the appropriate number of any ofthese packaged forms. It may be provided in single dose injectable form,for example in the form of a pen. Compositions may be formulated for anysuitable route and means of administration. Subcutaneous or transdermalmodes of administration may be particularly suitable for pharmaceuticalcompositions and therapeutic molecules described herein.

Therapeutic compositions are typically sterile and stable under theconditions of manufacture and storage. The composition may be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier may be a solvent ordispersion medium containing, for example, water, alcohol such asethanol, polyol (e.g. glycerol, propylene glycol, and liquidpolyethylene glycol), or any suitable mixtures. The proper fluidity maybe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by use of surfactants according to formulation chemistry well knownin the art. In certain embodiments, isotonic agents, e.g. sugars,polyalcohols such as mannitol, sorbitol, or sodium chloride may bedesirable in the composition. Prolonged absorption of injectablecompositions may be brought about by including in the composition anagent that delays absorption for example, monostearate salts andgelatin.

Solutions or suspensions used for intradermal or subcutaneousapplication typically include one or more of: a sterile diluent such aswater for injection, saline solution, fixed oils, polyethylene glycols,glycerine, propylene glycol or other synthetic solvents, antibacterialagents such as benzyl alcohol or methyl parabens: antioxidants such asascorbic acid or sodium bisulfite; chelating agents such asethylenediaminetetraacetic acid: buffers such as acetates, citrates orphosphates; and tonicity adjusting agents such as, e.g., sodium chlorideor dextrose. The pH can be adjusted with acids or bases, such ashydrochloric acid or sodium hydroxide, or buffers with citrate,phosphate, acetate and the like. Such preparations may be enclosed inampoules, disposable syringes or multiple dose vials made of glass orplastic.

Sterile injectable solutions may be prepared by incorporating acell-targeting molecule of the present invention in the required amountin an appropriate solvent with one or a combination of ingredientsdescribed above, as required, followed by sterilization microfiltration.Dispersions may be prepared by incorporating the active compound into asterile vehicle that contains a dispersion medium and other ingredients,such as those described above. In the case of sterile powders for thepreparation of sterile injectable solutions, the methods of preparationare vacuum drying and freeze-drying (lyophilization) that yield a powderof the active ingredient in addition to any additional desiredingredient from a sterile-filtered solution thereof.

When a therapeutically effective amount of a cell-targeting molecule ofthe present invention is designed to be administered by, e.g.intravenous, cutaneous or subcutaneous injection, the binding agent willbe in the form of a pyrogen-free, parenterally acceptable aqueoussolution. Methods for preparing parenterally acceptable proteinsolutions, taking into consideration appropriate pH, isotonicity,stability, and the like, are within the skill in the art. A preferredpharmaceutical composition for intravenous, cutaneous, or subcutaneousinjection will contain, in addition to binding agents, an isotonicvehicle such as sodium chloride injection, Ringer's injection, dextroseinjection, dextrose and sodium chloride injection, lactated Ringer'sinjection, or other vehicle as known in the art. A pharmaceuticalcomposition of the present invention may also contain stabilizers,preservatives, buffers, antioxidants, or other additives well known tothose of skill in the art.

As described elsewhere herein, a cell-targeting molecule of the presentinvention or composition thereof (e.g. pharmaceutical or diagnosticcomposition) may be prepared with carriers that will protect thecell-targeting molecule of the invention against rapid release, such asa controlled release formulation, including implants, transdermalpatches, and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Many methods for the preparation of such formulationsare patented or generally known to those skilled in the art (see e.g.Sustained and Controlled Release Drug Delivery Systems (Robinson J, ed.Marcel Dekker, Inc., NY, U.S., 1978).

In certain embodiments, the composition of the present invention (e.g.pharmaceutical or diagnostic compositions) may be formulated to ensure adesired distribution in vivo. For example, the blood-brain barrierexcludes many large and/or hydrophilic compounds. To target atherapeutic molecule or composition of the present invention to aparticular in viwo location, it can be formulated, for example, inliposomes which may comprise one or more moieties that are selectivelytransported into specific cells or organs, thus enhancing targeted drugdelivery. Exemplary targeting moieties include folate or biotin:mannosides; antibodies; surfactant protein A receptor: p120 catenin andthe like.

Pharmaceutical compositions include parenteral formulations designed tobe used as implants or particulate systems. Examples of implants aredepot formulations composed of polymeric or hydrophobic components suchas emulsions, ion exchange resins, and soluble salt solutions. Examplesof particulate systems are microspheres, microparticles, nanocapsules,nanospheres, and nanoparticles. Controlled release formulations may beprepared using polymers sensitive to ions, such as, e.g. liposomes,polaxamer 407, and hydroxyapatite.

Pharmaceutical compositions of the present invention may be producedusing techniques known in the art such that the produced compositionscomprise emulsions, liposomes, niosomes, polymeric nanoparticles, and/orsolid lipid nanoparticles (SLNs) (see e.g. Lakshmi P et al., VenerealLeprol 73: 157-161 (2007): A Revolution in Dosage Form Design andDevelopment, Recent Advances in Novel Drug Carrier Systems (Sezer A,ed., InTech, 2012)).

Diagnostic compositions of the present invention comprise acell-targeting molecule of the invention and one or more detectionpromoting agents. Various detection promoting agents are known in theart, such as isotopes, dyes, colorimetric agents, contrast enhancingagents, fluorescent agents, bioluminescent agents, and magnetic agents.These agents may be incorporated into the cell-targeting molecule of theinvention at any suitable position so long as requisite a functionalactivity(s) is retained. For example, the linkage or incorporation ofthe detection promoting agent may be via an amino acid residue(s) of thecell-targeting molecule of the present invention or via some type oflinkage known in the art, including via linkers and/or chelators. Theassociation of the detection promoting agent with a cell-targetingmolecule of a diagnostic composition of the present invention is in sucha way to enable the detection of the presence of the cell-targetingmolecule and/or its target cell after internalization of thecell-targeting molecule in a screen, assay, diagnostic procedure, and/orimaging technique.

There are numerous detection promoting agents known to the skilledworker which can be operably associated or linked to the cell-targetingmolecule of the present invention for information gathering methods,such as for diagnostic and/or prognostic applications to diseases,disorders, or conditions of an organism. For example, detectionpromoting agents include image enhancing contrast agents, such asfluorescent dyes (e.g. Alexa680, indocyanine green, and Cy5.5), isotopesand radionuclides, such as ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ³²P, ⁵¹Mn, ⁵²mMn, ⁵²Fe,⁵⁵Co, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁷³Se, ⁷⁵Br, ⁷⁶Br, ⁸²mRb, ⁸³Sr,⁸⁶Y, ⁹⁰Y, ⁸⁹Zr, ⁹⁴mTc, ⁹⁴Tc ⁹⁹mTc, ¹¹⁰In, ¹¹¹In, ¹²⁰I, ¹²³I, ¹²⁴I, ¹²⁵I,¹³¹I, ¹⁵⁴Gd, ¹⁵⁵Gd, ¹⁵⁶Gd, ¹⁵⁷Gd, ¹⁵⁸Gd, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, and ²²³Rparamagnetic ions, such as chromium (III), manganese (II), iron (III),iron (II), cobalt (II), nickel (II), copper (II), neodymium (III),samarium (III), ytterbium (III), gadolinium (III), vanadium (II),terbium (III), dysprosium (III), holmium (III) or erbium (III); metals,such as lanthanum (III), gold (III), lead (II), and bismuth (III);ultrasound-contrast enhancing agents, such as liposomes; radiopaqueagents, such as barium, gallium, and thallium compounds. Detectionpromoting agents may be incorporated directly or indirectly by using anintermediary functional group, such as chelators like 2-benzyl DTPA,PAMAM, NOTA, DOTA, TETA, analogs thereof, and functional equivalents ofany of the foregoing.

There are numerous imaging approaches in the art which are known to theskilled worker, such as non-invasive in vivo imaging techniques commonlyused in the medical arena, for example: computed tomography imaging (CTscanning), optical imaging (including direct, fluorescent, andbioluminescent imaging), magnetic resonance imaging (MRI), positronemission tomography (PET), single-photon emission computed tomography(SPECT), ultrasound, and x-ray computed tomography imaging.

The phrase “diagnostically sufficient amount” refers to an amount thatprovides adequate detection and accurate measurement for informationgathering purposes by the particular assay or diagnostic techniqueutilized. Generally, the diagnostically sufficient amount for wholeorganism, in vivo, diagnostic use will be a non-cumulative dose ofbetween 0.001 mg to 1 mg of the detection promoting agent linked tocell-targeting molecule per kilogram (kg) of subject per subject(mg/kg). However, the diagnostically sufficient amount for wholeorganism, in vivo, diagnostic use may be a non-cumulative dose ofbetween 0.0001 mg to 10 mg of the detection promoting agent linked tocell-targeting molecule per kilogram (kg) of subject per subject(mg/kg). Typically, the amount of cell-targeting molecule of the presentinvention used in these information-gathering methods will be as low aspossible provided that it is still a diagnostically sufficient amount.For example, for in vivo detection in an organism, the amount ofcell-targeting molecule or diagnostic composition of the presentinvention administered to a subject will be as low as feasibly possible.

VII. Production or Manufacture of a Pharmaceutical and/or DiagnosticComposition Comprising a Cell-Targeting Molecule of the PresentInvention

Pharmaceutically acceptable salts or solvates of any of thecell-targeting molecules of the invention are likewise within the scopeof the present invention.

The term “solvate” in the context of the present invention refers to acomplex of defined stoichiometry formed between a solute (in casu, acell-targeting molecule or pharmaceutically acceptable salt thereofaccording to the invention) and a solvent. The solvent in thisconnection may, for example, be water, ethanol or anotherpharmaceutically acceptable, typically small-molecular organic species,such as, but not limited to, acetic acid or lactic acid. When thesolvent in question is water, such a solvate is normally referred to asa hydrate.

Cell-targeting molecules of the present invention, or salts thereof, maybe formulated as pharmaceutical compositions prepared for storage oradministration, which typically comprise a therapeutically effectiveamount of a compound of the present invention, or a salt thereof in apharmaceutically acceptable carrier. The term “pharmaceuticallyacceptable carrier” includes any of the standard pharmaceuticalcarriers. Pharmaceutically acceptable carriers for therapeutic use arewell known in the pharmaceutical art, and are described, for example, inRemington's Pharmaceutical Sciences (Mack Publishing Co. (A. Gennaro,ed., 1985)). As used herein, “pharmaceutically acceptable carrier”includes any and all physiologically acceptable, i.e. compatible,solvents, dispersion media, coatings, antimicrobial agents, isotonic,and absorption delaying agents, and the like. Pharmaceuticallyacceptable carriers or diluents include those used in formulationssuitable for oral, rectal, nasal or parenteral (including subcutaneous,intramuscular, intravenous, intradermal, and transdermal)administration. Exemplary pharmaceutically acceptable carriers includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. Examples of suitable aqueous and nonaqueous carriers thatmay be employed in the pharmaceutical compositions of the inventioninclude water, ethanol, polyols (such as glycerol propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyloleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants. In certain embodiments, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g. by injection or infusion). Depending onselected route of administration, the protein or other pharmaceuticalcomponent may be coated in a material intended to protect the compoundfrom the action of low pH and other natural inactivating conditions towhich the active protein may encounter when administered to a patient bya particular route of administration.

The formulations of the pharmaceutical compositions of the invention mayconveniently be presented in unit dosage form and may be prepared by anyof the methods well known in the art of pharmacy. In such form, thecomposition is divided into unit doses containing appropriate quantitiesof the active component. The unit dosage form can be a packagedpreparation, the package containing discrete quantities of thepreparations, for example, packeted tablets, capsules, and powders invials or ampoules. The unit dosage form can also be a capsule, cachet,or tablet itself, or it can be the appropriate number of any of thesepackaged forms. It may be provided in single dose injectable form, forexample in the form of a pen. Compositions may be formulated for anysuitable route and means of administration. Subcutaneous or transdermalmodes of administration may be particularly suitable for pharmaceuticalcompositions and therapeutic molecules described herein.

The pharmaceutical compositions of the present invention may alsocontain adjuvants such as preservatives, wetting agents, emulsifyingagents and dispersing agents. Preventing the presence of microorganismsmay be ensured both by sterilization procedures, and by the inclusion ofvarious antibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. Isotonic agents, suchas sugars, sodium chloride, and the like into the compositions, may alsobe desirable. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption such as, aluminum monostearate and gelatin.

A pharmaceutical composition of the present invention also optionallyincludes a pharmaceutically acceptable antioxidant. Exemplarypharmaceutically acceptable antioxidants are water soluble antioxidantssuch as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; oil-soluble antioxidants,such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylatedhydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and thelike: and metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, andthe like.

In another aspect, the present invention provides pharmaceuticalcompositions comprising one or a combination of different cell-targetingmolecules of the invention, or an ester, salt or amide of any of theforegoing, and at least one pharmaceutically acceptable carrier.

Therapeutic compositions are typically sterile and stable under theconditions of manufacture and storage. The composition may be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier may be a solvent ordispersion medium containing, for example, water, alcohol such asethanol, polyol (e.g. glycerol, propylene glycol, and liquidpolyethylene glycol), or any suitable mixtures. The proper fluidity maybe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by use of surfactants according to formulation chemistry well knownin the art. In certain embodiments, isotonic agents, e.g. sugars,polyalcohols such as mannitol, sorbitol, or sodium chloride may bedesirable in the composition. Prolonged absorption of injectablecompositions may be brought about by including in the composition anagent that delays absorption for example, monostearate salts andgelatin.

Solutions or suspensions used for intradermal or subcutaneousapplication typically include one or more of: a sterile diluent such aswater for injection, saline solution, fixed oils, polyethylene glycols,glycerine, propylene glycol or other synthetic solvents: antibacterialagents such as benzyl alcohol or methyl parabens; antioxidants such asascorbic acid or sodium bisulfite; chelating agents such asethylenediaminetetraacetic acid; buffers such as acetates, citrates orphosphates; and tonicity adjusting agents such as, e.g., sodium chlorideor dextrose. The pH can be adjusted with acids or bases, such ashydrochloric acid or sodium hydroxide, or buffers with citrate,phosphate, acetate and the like. Such preparations may be enclosed inampoules, disposable syringes or multiple dose vials made of glass orplastic.

Sterile injectable solutions may be prepared by incorporating acell-targeting molecule of the invention in the required amount in anappropriate solvent with one or a combination of ingredients describedabove, as required, followed by sterilization microfiltration.Dispersions may be prepared by incorporating the active compound into asterile vehicle that contains a dispersion medium and other ingredients,such as those described above. In the case of sterile powders for thepreparation of sterile injectable solutions, the methods of preparationare vacuum drying and freeze-drying (lyophilization) that yield a powderof the active ingredient in addition to any additional desiredingredient from a sterile-filtered solution thereof.

When a therapeutically effective amount of a cell-targeting molecule ofthe invention is designed to be administered by, e.g. intravenous,cutaneous or subcutaneous injection, the binding agent will be in theform of a pyrogen-free, parenterally acceptable aqueous solution.Methods for preparing parenterally acceptable protein solutions, takinginto consideration appropriate pH, isotonicity, stability, and the like,are within the skill in the art. A preferred pharmaceutical compositionfor intravenous, cutaneous, or subcutaneous injection will contain, inaddition to binding agents, an isotonic vehicle such as sodium chlorideinjection, Ringer's injection, dextrose injection, dextrose and sodiumchloride injection, lactated Ringer's injection, or another vehicle asknown in the art. A pharmaceutical composition of the present inventionmay also contain stabilizers, preservatives, buffers, antioxidants, orother additives well known to those of skill in the art.

As described elsewhere herein, a cell-targeting molecule, or compositionof the present invention (e.g. pharmaceutical or diagnostic composition)may be prepared with carriers that will protect the cell-targetingmolecule against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art (see e.g. Sustained andControlled Release Drug Delivery Systems (Robinson J, ed., MarcelDekker, Inc., NY, U.S., 1978)).

In certain embodiments, the composition of the present invention (e.g.pharmaceutical or diagnostic composition) may be formulated to ensure adesired distribution in vivo. For example, the blood-brain barrierexcludes many large and/or hydrophilic compounds. To target atherapeutic cell-targeting molecule or composition of the invention to aparticular in vivo location, it can be formulated, for example, inliposomes which may comprise one or more moieties that are selectivelytransported into specific cells or organs, thus enhancing targeted drugdelivery. Exemplary targeting moieties include folate or biotin;mannosides; antibodies; surfactant protein A receptor; p120 catenin andthe like.

Pharmaceutical compositions include parenteral formulations designed tobe used as implants or particulate systems. Examples of implants aredepot formulations composed of polymeric or hydrophobic components suchas emulsions, ion exchange resins, and soluble salt solutions. Examplesof particulate systems are microspheres, microparticles, nanocapsules,nanospheres, and nanoparticles (see e.g. Honda M et al., Int JNanomedicine 8: 495-503 (2013); Sharma A et al., Biomed Res Int 2013:960821 (2013); Ramishetti S, Huang L, Ther Deliv 3: 1429-45 (2012)).Controlled release formulations may be prepared using polymers sensitiveto ions, such as, e.g. liposomes, polaxamer 407, and hydroxyapatite.

VIII. Polynucleotides, Expression Vectors, and Host Cells of theInvention

Beyond the cell-targeting molecules of the present invention and theirpolypeptide components, the polynucleotides that encode the polypeptidesand cell-targeting molecules of the invention, or functional portionsthereof, are also encompassed within the scope of the present invention.The term “polynucleotide” is equivalent to the term “nucleic acid,” eachof which includes one or more of: polymers of deoxyribonucleic acids(DNAs), polymers of ribonucleic acids (RNAs), analogs of these DNAs orRNAs generated using nucleotide analogs, and derivatives, fragments andhomologs thereof. The polynucleotide of the present invention may besingle-, double-, or triple-stranded. Such polynucleotides arespecifically disclosed to include all polynucleotides capable ofencoding an exemplary protein, for example, taking into account thewobble known to be tolerated in the third position of RNA codons, yetencoding for the same amino acid as a different RNA codon (see StothardP, Biotechniques 28: 1102-4 (2000)).

In one aspect, the invention provides polynucleotides which encode acell-targeting molecule of the invention (e.g. a fusion protein), or apolypeptide fragment or derivative thereof. The polynucleotides mayinclude, e.g., nucleic acid sequence encoding a polypeptide of at least50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more, identityto a polypeptide comprising one of the amino acid sequences of theprotein. The invention also includes polynucleotides comprisingnucleotide sequences that hybridize under stringent conditions to apolynucleotide which encodes a cell-targeting molecule of the invention,or a polypeptide fragment or derivative thereof or the antisense orcomplement of any such sequence.

Derivatives or analogs of the cell-targeting molecules of the presentinvention include, inter alia, polynucleotide (or polypeptide) moleculeshaving regions that are substantially homologous to the polynucleotides,cell-targeting molecules, or polypeptide components of thecell-targeting molecules of the present invention, e.g. by at leastabout 45%, 50%, 70%, 80%, 95%, 98%, or even 99% identity (with apreferred identity of 80-99%) over a polynucleotide or polypeptidesequence of the same size or when compared to an aligned sequence inwhich the alignment is done by a computer homology program known in theart. An exemplary program is the GAP program (Wisconsin SequenceAnalysis Package, Version 8 for UNIX, Genetics Computer Group,University Research Park, Madison, Wis., U.S.) using the defaultsettings, which uses the algorithm of Smith T. Waterman M, Adv Appl Math2: 482-9 (1981). Also included are polynucleotides capable ofhybridizing to the complement of a sequence encoding the cell-targetingmolecule of the invention under stringent conditions (see e.g. Ausubel Fet al., Current Protocols in Molecular Biology (John Wiley & Sons, NewYork, N.Y. U.S., 1993)), and below. Stringent conditions are known tothose skilled in the art and may be found, e.g., in Current Protocols inMolecular Biology (John Wiley & Sons, NY, U.S., Ch. Sec. 6.3.1-6.3.6(1989)).

The present invention further provides expression vectors that comprisethe polynucleotides within the scope of the present invention. Thepolynucleotides capable of encoding the cell-targeting molecules of theinvention, or polypeptide components thereof, may be inserted into knownvectors, including bacterial plasmids, viral vectors and phage vectors,using material and methods well known in the art to produce expressionvectors. Such expression vectors will include the polynucleotidesnecessary to support production of contemplated cell-targeting moleculesof the invention within any host cell of choice or cell-free expressionsystems (e.g. pTxb1 and pIVEX2.3). The specific polynucleotidescomprising expression vectors for use with specific types of host cellsor cell-free expression systems are well known to one of ordinary skillin the art, can be determined using routine experimentation, or may bepurchased.

The term “expression vector,” as used herein, refers to apolynucleotide, linear or circular, comprising one or more expressionunits. The term “expression unit” denotes a polynucleotide segmentencoding a polypeptide of interest and capable of providing expressionof the nucleic acid segment in a host cell. An expression unit typicallycomprises a transcription promoter, an open reading frame encoding thepolypeptide of interest, and a transcription terminator, all in operableconfiguration. An expression vector contains one or more expressionunits. Thus, in the context of the present invention, an expressionvector encoding a cell-targeting molecule of the invention (e.g. a scFvgenetically recombined with a Shiga toxin effector polypeptide fused toa T-cell epitope-peptide) includes at least an expression unit for thesingle polypeptide chain, whereas a protein comprising, e.g. two or morepolypeptide chains (e.g. one chain comprising a V_(L) domain and asecond chain comprising a V_(H) domain linked to a toxin effectorregion) includes at least two expression units, one for each of the twopolypeptide chains of the protein. For expression of multi-chaincell-targeting proteins of the invention, an expression unit for eachpolypeptide chain may also be separately contained on differentexpression vectors (e.g. expression may be achieved with a single hostcell into which expression vectors for each polypeptide chain has beenintroduced).

Expression vectors capable of directing transient or stable expressionof polypeptides and proteins are well known in the art. The expressionvectors generally include, but are not limited to, one or more of thefollowing: a heterologous signal sequence or peptide, an origin ofreplication, one or more marker genes, an enhancer element, a promoter,and a transcription termination sequence, each of which is well known inthe art. Optional regulatory control sequences, integration sequences,and useful markers that can be employed are known in the art.

Cell-free systems can be used to produce cell-targeting molecules of thepresent invention (see e.g. Jaing X et al., FEBS Lett 514: 290-4 (2002);Kawasaki T et al., Eur J Biochem 270: 4780-6 (2003); Ali M et al., JBiosci Bioeng 99: 181-6 (2005); Galeffi P et al., J Transl Med 4: 39(2006); Han Y et al., Biotechnol Prog 22: 1084-9 (2006): Schenk J etal., Biochimie 89: 1304-11 (2007): Oh I et al., Bioproc Biosyst Eng 33127-32 (2010): Merk H et al., BioTechniques 53: 153-60 (2012); Stech Met al., J Biotechnol 164: 220-31 (2012); Yin G et al., mAbs 4:217-(2012); GroffD et al., mAbs 6: 671-8 (2014): Stech M et al., EngLife Sci 14: 387-98 (2014): Stech M, Kubick S, Antibodies 4: 12-33(2015); Thoring L et al., Sci Rep 7: 11710 (2017); Stech M et al., SciRep 7: 12030 (2017)).

The term “host cell” refers to a cell which can support the replicationor expression of the expression vector. Host cells may be prokaryoticcells, such as E, coli or eukaryotic cells (e.g. yeast, insect,amphibian, bird, or mammalian cells). Creation and isolation of hostcell lines comprising a polynucleotide of the invention or capable ofproducing a cell-targeting molecule of the invention, or polypeptidecomponent thereof, can be accomplished using standard techniques knownin the art.

Cell-targeting molecules within the scope of the present invention maybe variants or derivatives of the polypeptides and proteins describedherein that are produced by modifying the polynucleotide encoding apolypeptide and/or protein by altering one or more amino acids ordeleting or inserting one or more amino acids that may render it moresuitable to achieve desired properties, such as more optimal expressionby a host cell.

IX. Delivery Devices and Kits

In certain embodiments, the invention relates to a device comprising oneor more compositions of matter of the invention, such as apharmaceutical composition, for delivery to a subject in need thereof.Thus, a delivery device comprising one or more compounds of theinvention may be used to administer to a patient a composition of matterof the invention by various delivery methods, including: intravenous,subcutaneous, intramuscular or intraperitoneal injection: oraladministration: transdermal administration; pulmonary or transmucosaladministration: administration by implant, osmotic pump, cartridge ormicro pump; or by other means recognized by a person of skill in theart.

Also within the scope of the present invention are kits comprising atleast one composition of matter of the invention, and optionally,packaging and instructions for use. Kits may be useful for drugadministration and/or diagnostic information gathering. A kit of theinvention may optionally comprise at least one additional reagent (e.g.,standards, markers and the like). Kits typically include a labelindicating the intended use of the contents of the kit. The kit mayfurther comprise reagents and other tools for detecting a cell-type(e.g. a tumor cell) in a sample or in a subject, or for diagnosingwhether a patient belongs to a group that responds to a therapeuticstrategy which makes use of a cell-targeting molecule of the presentinvention, or composition thereof, or related method of the presentinvention as described herein.

X. Methods for Using a Cell-Targeting Molecule of the Present Inventionand Pharmaceutical Composition and/or Diagnostic Composition Thereof

Generally, it is an object of the present invention to providepharmacologically active agents, as well as compositions comprising thesame, that can be used in the prevention and/or treatment of diseases,disorders, and conditions, such as certain cancers, tumors, growthabnormalities, immune disorders, or further pathological conditionsmentioned herein. Accordingly, the present invention provides methods ofusing the cell-targeting molecules, pharmaceutical compositions, anddiagnostic compositions of the present invention for the delivery of aCD8+ T-cell epitope-peptide cargo to the MHC class I presentationpathways of target cells, targeted killing of specific cells, labelingof the cell-surfaces of target cells with specific pMHC Is and/orspecific interior compartments of target cells, for collectingdiagnostic information, and for treating diseases, disorders, andconditions as described herein. For example, the methods of the presentinvention may be used as an immunotherapy to prevent or treat cancers,cancer initiation, tumor initiation, metastasis, and/or cancer diseasereoccurrence.

In particular, it is an object of the present invention to provide suchpharmacologically active agents, compositions, and/or methods that havecertain advantages compared to the agents, compositions, and/or methodsthat are currently known in the art. Accordingly, the present inventionprovides methods of using cell-targeting molecules characterized byspecified protein sequences and pharmaceutical compositions thereof. Forexample, any of the polypeptide sequences in SEQ ID NOs: 4-255, 259-278,and 288-748 may be specifically utilized as a component of thecell-targeting molecules used in the following methods or any method forusing a cell-targeting molecule known to the skilled worker, such as,e.g., various methods described in WO 2014/164680, WO 2014/164693, WO2015/138435, WO 2015/138452, WO 2015/113005, WO 2015/113007. WO2015/191764, US2015/259428, US2014/965882, WO 2016/196344, WO2017/019623, and WO 2018/106895.

The present invention provides methods of delivering a CD8+ T-cellepitope-peptide cargo to a cell, the method comprising the step ofcontacting the cell, either in vitro or in vivo, with a cell-targetingmolecule or pharmaceutical composition of the present invention. Incertain further embodiments, the cell-targeting molecule of the presentinvention causes, after the contacting step, an intercellular engagementof the cell by an immune cell, such as, e.g., a CD8+ T-cell and/or CTL,either in vitro cell culture or in vivo within a living chordate. Thepresentation of a CD8+ T-cell epitope by a target cell within anorganism can lead to the activation of robust immune responses to atarget cell and/or its general locale within an organism. Thus, thetargeted delivery of a CD8+ T-cell epitope cargo for presentation may beutilized for as a mechanism for activating CD8+ T-cell responses duringa therapeutic regime and/or vaccination strategy.

The present invention provides methods of delivering to a MHC class 1presentation pathway of a chordate cell a CD8+ T-cell epitope-peptide,the method comprising the step of contacting the cell either in vitro orin vivo, with a cell-targeting molecule, pharmaceutical composition,and/or diagnostic composition of the present invention. In certainfurther embodiments, the cell-targeting molecule of the presentinvention causes, after the contacting step, an intercellular engagementof the cell by an immune cell, such as, e.g., a CD8+ T-cell and/or CTLeither in vitro cell culture or in vivo within a chordate.

The delivery of the CD8+ T-cell epitope-peptide cargo to the MHC class Ipresentation pathway of a target cell using a cell-targeting molecule ofthe invention can be used to induce the target cell to present theepitope-peptide in association with MHC class I molecules on a cellsurface. In a chordate, the presentation of an immunogenic, CD8+ T-cellepitope by the MHC class I complex can sensitize the presenting cell forkilling by CTL-mediated cytolysis, induce immune cells into altering themicroenvironment, and signal for the recruitment of more immune cells tothe target cell site within the chordate. Thus, the cell-targetingmolecules of the present invention, and compositions thereof, can beused to kill a specific cell-type upon contacting a cell or cells with acell-targeting molecule of the present invention and/or can be used tostimulate an immune response in a chordate.

By engineering MHC class I epitopes, such as, e.g., from a known viralantigen, into cell-targeting molecules, the targeted delivery andpresentation of immuno-stimulatory antigens may be used to harness anddirect beneficial function(s) of a chordate immune cell, e.g. in vitro,and/or a chordate immune system in vivo. This may be accomplished byexogenous administration of the cell-targeting molecule into anextracellular space, such as, e.g., the lumen of a blood vessel, andthen allowing for the cell-targeting molecule to find a target cell,enter the cell, and intracellularly deliver its CD8+ T-cell epitopecargo. The applications of these CD8+ T-cell epitope cargo delivery andMHC class I presenting functions of the cell-targeting molecules of thepresent invention are vast. For example, the delivery of a CD8+ epitopecargo to a cell and the MHC class I presentation of the deliveredepitope by the cell in a chordate can cause the intercellular engagementof a CD8+ effector T-cell and may lead to a CTL(s) killing the targetcell and/or secreting immuno-stimulatory cytokines.

Certain embodiments of the present invention is an immunotherapeuticmethod, the method comprising the step of administering to a patient, inneed thereof, a cell-targeting molecule and/or pharmaceuticalcomposition of the present invention. In certain further embodiments,the immunotherapeutic method is a method of treating a disease,disorder, and/or condition (such as, e.g., a cancer, tumor, growthabnormality, immune disorder, and/or microbial infection), bystimulating a beneficial immune response in the patient.

Certain embodiments of the present invention is an immunotherapeuticmethod of treating cancer, the method comprising the step ofadministering to a patient, in need thereof, a cell-targeting moleculeand/or pharmaceutical composition of the present invention. In certainfurther embodiments, the method comprises the additional steps ofadministering to the patient an adjuvant and/or microbiome-alteringagent (see e.g. Vetizou M et al., Science 350: 1079-84 (2015); Ayelet Set al., Science 350: 1084-9 (2015): US2015/352206; WO2016/063263).

The present invention provides immunotherapy methods involvingdelivering a CD8+ T-cell epitope-peptide cargo to a target cell in achordate and causing an immune response, the method comprising the stepof administering to the chordate a cell-targeting molecule orpharmaceutical composition of the present invention. For certain furtherembodiments, the immune response is an intercellular immune cellresponse selected from the group consisting of: CD8+ immune cellsecretion of a cytokine(s), CTL induced growth arrest in the targetcell, CTL induced necrosis of the target cell, CTL induced apoptosis ofthe target cell, non-specific cell death in a tissue locus,intermolecular epitope spreading, breaking immunological tolerance to amalignant cell type, and the chordate acquiring persistent immunity to amalignant cell-type (see e.g. Matsushita H et al., Cancer Immunol Res 3:26-36 (2015)). These immune responses can be detected and/or quantifiedusing techniques known to the skilled worker. For example, CD8+ immunecells can release immuno-stimulatory cytokines, such as, e.g., IFN-γ,tumor necrosis factor alpha (TNFα), macrophage inflammatory protein-1beta (MIP-1β), and interleukins such as IL-17, IL-4, IL-22, and IL-2(see e.g. Examples, infra; Seder R et al., Nat Rev Immunol 8: 247-58(2008)). IFN-γ can increase MHC class I molecule expression andsensitize neoplastic cells to CTL-mediated cell killing (Vlkova V etal., Oncotarget 5: 6923-35 (2014)). Inflammatory cytokines can stimulatebystander T-cells that harbor unrelated TCR specificities to thecytokine releasing cell (see e.g. Tough D et al., Science 272; 1947-50(1996)). Activated CTLs can indiscriminately kill cells proximal toepitope-MHC class I complex presenting cell regardless of the proximalcell's present peptide-MHC class I complex repertoire (Wiedemann A etal., Proc Natl Acad Sci USA 103: 10985-90 (2006)). Thus, for certainfurther embodiments, the immune response is an intercellular immune cellresponse selected from the group consisting of: proximal cell killingmediated by immune cells where the proximal cell is not displaying anyCD8+ T-cell epitope-peptide delivered by the cell-targeting molecule ofthe present invention and regardless of the presence of anyextracellular target biomolecule of the binding region of thecell-targeting molecule physically coupled to the proximal cell(s) thatis killed.

The presence of non-self epitopes in CTL-lysed cells, whether targetcells or cells merely proximal to target cells, can be recognized andtargeted as foreign by the immune system, including recognition ofnon-self epitopes in target cells via the mechanism of intermolecularepitope spreading (see McCluskey J et al., Immunol Rev 164: 209-29(1998); Vanderlugt C et al., Immunol Rev 164: 63-72 (1998); VanderlugtC, Miller S, Nat Rev Immunol 2: 85-95 (2002)). Proximal cells mayinclude non-neoplastic cells, such as, e.g., cancer associatedfibroblasts, mesenchymal stem cells, tumor-associated endothelial cells,and immature myeloid-derived suppressor cells. For example, a cancercell may harbor on average 25 to 500 nonsynonymous mutations in codingsequences (see e.g. Fritsch E et al., Cancer Immunol Res 2: 522-9(2014)). Both cancer driver and non-driver mutations are part of themutational landscape of a cancer cell that corresponds to numerousnon-self epitopes per cell and the average tumor may possess ten or morenon-self epitopes (see e.g. Segal N et al., Cancer Res 68: 889-92(2008)). For example, mutant forms of the tumor protein p53 can containnon-self epitopes (see e.g. Vigneron N et al., Cancer Immun 13: 15(2013)). In addition, the presence of non-self epitopes, such as mutatedself-proteins, can result in the production of memory cells specific tothose new epitope(s). Because certain embodiments of the cell-targetingmolecules of the present invention may increase dendritic cell samplingat a targeted tissue locus, the probability of cross-priming the immunesystem with intracellular antigens may be increased (see e.g. Chiang Cet al., Expert Opin Biol Ther 15: 569-82 (2015)). Thus, as a result ofcell-targeting molecule delivery of a heterologous, CD8+ T-cell epitopecargo and MHC class I presentation of that epitope, target cells andother proximal cells containing non-self epitopes can be rejected by theimmune system, including via non-self epitopes other than epitopesdelivered by a cell-targeting molecule of the invention. Such mechanismscould, e.g., induce antitumor immunity against tumor cells which do notexpress the extracellular target biomolecule of the binding region ofthe cell-targeting molecule.

Immune responses which involve cytokine secretion and/or T-cellactivation may result in modulation of the immuno-microenvironment of alocus within a chordate. A method of the present invention may be usedto alter the microenvironment of a tissue locus within a chordate inorder to change the regulatory homeostasis on immune cells, such as,e.g. tumor-associated macrophages, T-cells. T helper cells, antigenpresenting cells, and natural killer cells.

For certain embodiments, a method of the present invention may be usedto enhance anti-tumor cell immunity in a chordate subject and/or tocreate a persistent anti-tumor immunity in a chordate, such as, e.g.,due to the development of memory T-cells and/or alterations to the tumormicroenvironment.

Certain embodiments of the cell-targeting molecules of the presentinvention, or pharmaceutical compositions thereof, can be used to “seed”a locus within a chordate with non-self, CD8+ T-cell epitope-peptidepresenting cells in order to stimulate the immune system to police thelocus with greater strength and/or to alleviate immuno-inhibitorysignals, e.g., anergy inducing signals. In certain further embodimentsof this “seeding” method of the present invention, the locus is a tumormass or infected tissue site. In certain embodiments of this “seeding”method of the present invention, the non-self, CD8+ T-cellepitope-peptide is selected from the group consisting of: peptides notalready presented by the target cells of the cell-targeting molecule,peptides not present within any protein expressed by the target cell,peptides not present within the proteome or transcriptome of the targetcell, peptides not present in the extracellular microenvironment of thesite to be seeded, and peptides not present in the tumor mass or infecttissue site to be targeting.

This “seeding” method functions to label one or more target cells withina chordate with one or more MHC class I presented CD8+ T-cell epitopes(pMHC Is) for intercellular recognition by immune cells and activationof downstream immune responses. By exploiting the cell-internalizing,intracellularly routing, and/or MHC class I epitope delivering functionsof the cell-targeting molecules of the present invention, the targetcells that display the delivered CD8+ T-cell epitope can be recognizedby immunosurveillance mechanisms of the chordate's immune cells andresult in intercellular engagement of the presenting target cell by CD8+T-cells, such as, e.g., CTLs. This “seeding” method of using acell-targeting molecule of the present invention may stimulate immunecell mediated killing of target cells regardless of whether they arepresenting a cell-targeting molecule-delivered T-cell epitope(s), suchas, e.g., as a result of intermolecular epitope spreading and/orbreaking of immuno-tolerance to the target cell based on presentation ofendogenous antigens as opposed to artificially delivered epitopes. This“seeding” method of using a cell-targeting molecule of the presentinvention may provide a vaccination effect (new epitope(s) exposure)and/or vaccination-booster-dose effect (epitope re-exposure) by inducingadaptive immune responses to cells within the seeded microenvironment,such as, e.g. a tumor mass or infected tissue site, based on thedetection of epitopes which are either recognized as foreign by naïveT-cells and/or already recognizable as non-self (i.e. recall antigens)by memory T-cells. This “seeding” method may also induce the breaking ofimmuno-tolerance to a target cell population, a tumor mass, diseasedtissue site, and/or infected tissue site within a chordate, eitherperipherally or systemically.

The presence of dying or necrotic tumor cells at site or loci within achordate may result in a localized immune stimulation effect. Forexample, dying or necrotic tumor cells can release factors, such as,e.g., high mobility group proteins and/or ATP, which in turn canstimulate immunogenic maturation of immune cells. The seeding of a tumorlocus may also induce or increase the ectopic expression of ER proteins(e.g., calreticulin) on the plasma membrane of tumor cells which in turncan promote/increase MHC class antigen presentation and phagocytosis oftumor cells at that site.

Certain methods of the present invention involving the seeding of alocus within a chordate with one or more antigenic and/or immunogenicCD8+ T-cell epitopes may be combined with the administration ofimmunologic adjuvants, whether administered locally or systemically, tostimulate the immune response to certain antigens, such as, e.g., theco-administration of a composition of the present invention with one ormore immunologic adjuvants like a cytokine, bacterial product, or plantsaponin. Other examples of immunologic adjuvants which may be suitablefor use in the methods of the present invention include aluminum saltsand oils, such as, e.g., alums, aluminum hydroxide, mineral oils,squalene, paraffin oils, peanut oils, and thimerosal.

Certain methods of the present invention involve promoting immunogeniccross-presentation and/or cross-priming of naïve CD8+ T-cells in achordate. For certain methods of the present invention, cross-primingoccurs as a result of the death, and/or the manner of death, of a targetcell caused by a cell-targeting molecule of the present invention suchthat the exposure of intracellular antigens in the dying or dead targetcell to immunosurveillance mechanisms is promoted.

Because multiple, heterologous, CD8+ T-cell epitopes (either as cargosor as embedded or inserted regions of a Shiga toxin effector polypeptidecomponent) may be delivered by a single cell-targeting molecule of thepresent invention, a single embodiment of the cell-targeting molecule ofthe present invention may be therapeutically effective in differentindividual chordates of the same species with different MHC I classmolecule variants, such as, e.g., in humans with different HLA alleles.This ability of certain embodiments of the present invention may allowfor the combining within a single cell-targeting molecule of differentCD8+ T-cell epitope-peptides with different therapeutic effectiveness indifferent sub-populations of subjects based on MHC class I moleculediversity and polymorphisms. For example, human MHC class I molecules,the HLA proteins, vary among humans based on genetic ancestry, e.g.African (sub-Saharan), Amerindian, Caucasoid, Mongoloid. New Guinean andAustralian, or Pacific islander.

Cell-targeting molecules of the present invention which can deliverheterologous, CD8+ T-cell epitopes from CMV antigens may be particularlyeffective because a majority of the human population has specific setsof CD8+ T-cells primed to react to CMV antigens and are constantlyrepressing chronic CMV infections to remain asymptomatic for theirentire life. In addition, elderly humans may reactive even more quicklyand strongly to CMV CD8+ T-cell epitopes due to age-related changes inthe adaptive immune system regarding CMV, such as, e.g., a potentiallymore focused immune surveillance toward CMV and as shown by thecomposition of the T-cell antigen receptor repertoire and relative CTLlevels in more elderly humans (see e.g. Koch S et al., Ann NY Acad Sci1114: 23-35 (2007): Vescovini R et al., J Immunol 184: 3242-9 (2010):Cicin-Sain L et al., J Immunol 187: 1722-32 (2011): Fillp T et al.,Front Immunol 4: 271 (2013): Pawelec G, Exp Gerontol 54: 1-5 (2014)).

The present invention provides methods of killing a cell comprising thestep of contacting the cell, either in vitro or in vivo, with acell-targeting molecule or pharmaceutical composition of the presentinvention. The cell-targeting molecules and pharmaceutical compositionsof the present invention can be used to kill a specific cell-type uponcontacting a cell or cells with one of the claimed compositions ofmatter. In certain embodiments, a cell-targeting molecule orpharmaceutical composition of the present invention can be used to killspecific cell-types in a mixture of different cell-types, such asmixtures comprising cancer cells, infected cells, and/or hematologicalcells. In certain embodiments, a cell-targeting molecule orpharmaceutical composition of the present invention can be used to killcancer cells in a mixture of different cell-types. In certainembodiments, a cell-targeting molecule or pharmaceutical composition ofthe present invention can be used to kill specific cell-types in amixture of different cell-types, such as pre-transplantation tissues. Incertain embodiments, a cell-targeting molecule or pharmaceuticalcomposition of the present invention can be used to kill specificcell-types in a mixture of cell-types, such as pre-administration tissuematerial for therapeutic purposes. In certain embodiments, acell-targeting molecule or pharmaceutical composition of the presentinvention can be used to selectively kill cells infected by viruses ormicroorganisms, or otherwise selectively kill cells expressing aparticular extracellular target biomolecule, such as a cell surfacebiomolecule. The cell-targeting molecules and pharmaceuticalcompositions of the present invention have varied applications,including, e.g., uses in depleting unwanted cell-types from tissueseither in vitro or in vivo, uses as antiviral agents, uses asanti-parasitic agents, and uses in purging transplantation tissues ofunwanted cell-types. In certain embodiments, a cell-targeting moleculeand/or pharmaceutical composition of the present invention can be usedto kill specific cell-types in a mixture of different cell-types, suchas pre-administration tissue material for therapeutic purposes, e.g.,pre-transplantation tissues. In certain embodiments, a cell-targetingmolecule or pharmaceutical composition of the present invention can beused to selectively kill cells infected by viruses or microorganisms, orotherwise selectively kill cells expressing a particular extracellulartarget biomolecule, such as a cell surface biomolecule.

The present invention provides a method of killing a cell in a patientin need thereof, the method comprising the step of administering to thepatient at least one cell-targeting molecule of the present invention ora pharmaceutical composition thereof. In certain embodiments of thecell-targeting molecule of the present invention, or pharmaceuticalcompositions thereof, can be used to kill an infected cell in a patientby targeting an extracellular biomolecule found physically coupled withan infected cell.

In certain embodiments, the cell-targeting molecule of the presentinvention or pharmaceutical compositions thereof can be used to kill acancer cell in a patient by targeting an extracellular biomolecule foundphysically coupled with a cancer or tumor cell. The terms “cancer cell”or “cancerous cell” refers to various neoplastic cells which grow anddivide in an abnormally accelerated and/or unregulated fashion and willbe clear to the skilled person. The term “tumor cell” includes bothmalignant and non-malignant cells. Generally, cancers and/or tumors canbe defined as diseases, disorders, or conditions that are amenable totreatment and/or prevention. The cancers and tumors (either malignant ornon-malignant) which are comprised of cancer cells and/or tumor cellswhich may benefit from methods and compositions of the invention will beclear to the skilled person. Neoplastic cells are often associated withone or more of the following: unregulated growth, lack ofdifferentiation, local tissue invasion, angiogenesis, and metastasis.The diseases, disorders, and conditions resulting from cancers and/ortumors (either malignant or non-malignant) which may benefit from themethods and compositions of the present invention targeting certaincancer cells and/or tumor cells will be clear to the skilled person.

Certain embodiments of the cell-targeting molecules and compositions ofthe present invention may be used to kill cancer stem cells, tumor stemcells, pre-malignant cancer-initiating cells, and tumor-initiatingcells, which commonly are slow dividing and resistant to cancertherapies like chemotherapy and radiation. For example, acute myeloidleukemias (AMLs) may be treated with the present invention by killingAML stem cells and/or dormant AML progenitor cells (see e.g. Shlush L etal., Blood 120: 603-12 (2012)). Cancer stem cells often overexpress cellsurface targets, such as, e.g., CD44, CD200, and others listed herein,which can be targets of certain binding regions of certain embodimentsof the cell-targeting molecules of the present invention (see e.g.Kawasaki B et al., Biochem Biophys Res Commun 364:778-82 (2007); Reim Fet al., Cancer Res 69: 8058-66 (2009)).

Because of the unique Shiga toxin A Subunit based mechanism of action,compositions of matter of the present invention may be more effectivelyused in methods involving their combination with, or in complementaryfashion with other therapies, such as, e.g., chemotherapies,immunotherapies, radiation, stem cell transplantation, and immunecheckpoint inhibitors, and/or effective againstchemoresistant/radiation-resistant and/or resting tumor cells/tumorinitiating cells/stem cells. Similarly, compositions of matter of thepresent invention may be more effectively used in methods involving incombination with other cell-targeted therapies targeting other than thesame epitope on, non-overlapping, or different targets for the samedisease disorder or condition.

Certain embodiments of the cell-targeting molecules of the presentinvention, or pharmaceutical compositions thereof, can be used to killan immune cell (whether healthy or malignant) in a patient by targetingan extracellular biomolecule found physically coupled with an immunecell.

It is within the scope of the present invention to utilize acell-targeting molecule of the present invention, or pharmaceuticalcomposition thereof, for the purposes of purging cell populations (e.g.bone marrow) of malignant and/or neoplastic cells and then reinfusingthe target-cell-depleted material into a patient in need thereof.

Additionally, the present invention provides a method of treating adisease, disorder, or condition in a patient comprising the step ofadministering to a patient in need thereof a therapeutically effectiveamount of at least one of the cell-targeting molecules of the presentinvention, or a pharmaceutical composition thereof. Contemplateddiseases, disorders, and conditions that can be treated using thismethod include cancers, malignant tumors, non-malignant tumors, growthabnormalities, immune disorders, and microbial infections.Administration of a “therapeutically effective dosage” of a compositionof the present invention can result in a decrease in severity of diseasesymptoms, an increase in frequency and duration of disease symptom-freeperiods, or a prevention of impairment or disability due to the diseaseaffliction.

The therapeutically effective amount of a composition of the presentinvention will depend on the route of administration, the type ofsubject being treated, and the physical characteristics of the specificpatient under consideration. These factors and their relationship todetermining this amount are well known to skilled practitioners in themedical arts. This amount and the method of administration can betailored to achieve optimal efficacy, and may depend on such factors asweight, diet, concurrent medication and other factors, well known tothose skilled in the medical arts. The dosage sizes and dosing regimenmost appropriate for human use may be guided by the results obtained bythe present invention, and may be confirmed in properly designedclinical trials. An effective dosage and treatment protocol may bedetermined by conventional means, starting with a low dose in laboratoryanimals and then increasing the dosage while monitoring the effects, andsystematically varying the dosage regimen as well. Numerous factors maybe taken into consideration by a clinician when determining an optimaldosage for a given subject. Such considerations are known to the skilledperson.

An acceptable route of administration may refer to any administrationpathway known in the art, including but not limited to aerosol, enteral,nasal, ophthalmic, oral, parenteral, rectal, vaginal, or transdermal(e.g. topical administration of a cream, gel or ointment, or by means ofa transdermal patch). “Parenteral administration” is typicallyassociated with injection at or in communication with the intended siteof action, including infraorbital, infusion, intraarterial,intracapsular, intracardiac, intradermal, intramuscular,intraperitoneal, intrapulmonary, intraspinal, intrastemal, intrathecal,intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous,transmucosal, or transtracheal administration.

For administration of a pharmaceutical composition of the presentinvention, the dosage range will generally be from about 0.001 to 10milligrams per kilogram (mg/kg), and more, usually 0.001 to 0.5 mg/kg,of the subject's body weight. Exemplary dosages may be 0.01 mg/kg bodyweight, 0.03 mg/kg body weight, 0.07 mg/kg body weight, 0.09 mg/kg bodyweight or 0.1 mg/kg body weight or within the range of 1-10 mg/kg. Anexemplary treatment regime is a once or twice daily administration, or aonce or twice weekly administration, once every two weeks, once everythree weeks, once every four weeks, once a month, once every two orthree months or once every three to 6 months. Dosages may be selectedand readjusted by the skilled health care professional as required tomaximize therapeutic benefit for a particular patient.

Pharmaceutical compositions of the present invention will typically beadministered to the same patient on multiple occasions. Intervalsbetween single dosages can be, for example, 2-5 days, weekly, monthly,every two or three months, every six months, or yearly. Intervalsbetween administrations can also be irregular, based on regulating bloodlevels or other markers in the subject or patient. Dosage regimens for acomposition of the present invention include intravenous administrationof 1 mg/kg body weight or 3 mg/kg body weight with the compositionadministered every two to four weeks for six dosages, then every threemonths at 3 mg/kg body weight or 1 mg/kg body weight.

A pharmaceutical composition of the present invention may beadministered via one or more routes of administration, using one or moreof a variety of methods known in the art. As will be appreciated by theskilled worker, the route and/or mode of administration will varydepending upon the desired results. Routes of administration forcell-targeting molecules, pharmaceutical compositions, and diagnosticcompositions of the present invention include, e.g. intravenous,intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, orother parenteral routes of administration, for example by injection orinfusion. For other embodiments, a cell-targeting molecules,pharmaceutical composition, and diagnostic composition of the presentinvention may be administered by a non-parenteral route, such as atopical, epidermal or mucosal route of administration, for example,intranasally, orally, vaginally, rectally, sublingually, or topically.

Therapeutic cell-targeting molecules of the present invention, orpharmaceutical compositions thereof, may be administered with one ormore of a variety of medical devices known in the art. For example, inone embodiment, a pharmaceutical composition of the invention may beadministered with a needleless hypodermic injection device. Examples ofwell-known implants and modules useful in the present invention are inthe art, including e.g., implantable micro-infusion pumps for controlledrate delivery; devices for administering through the skin; infusionpumps for delivery at a precise infusion rate; variable flow implantableinfusion devices for continuous drug delivery; and osmotic drug deliverysystems. These and other such implants, delivery systems, and modulesare known to those skilled in the art.

In certain embodiments, a cell-targeting molecule or pharmaceuticalcomposition of the present invention, alone or in combination with othercompounds or pharmaceutical compositions, can show potent cell-killactivity when administered to a population of cells, in vitro or in vivoin a subject such as in a patient in need of treatment. By targeting thedelivery of the Shiga toxin effector polypeptide associated with aheterologous CD8+ T-cell epitope cargo using high-affinity bindingregions to specific cell-types, Shiga toxin effector and/or CD8+ T-cellepitope presentation mediated cell-killing activities can be restrictedto specifically and selectively kill certain cell-types within anorganism, such as certain cancer cells, neoplastic cells, malignantcells, non-malignant tumor cells, or infected cells.

The cell-targeting molecule of the present invention, or pharmaceuticalcomposition thereof, may be administered alone or in combination withone or more other therapeutic or diagnostic agents. A combinationtherapy may include a cell-targeting molecule of the present invention,or pharmaceutical composition thereof, combined with at least one othertherapeutic agent selected based on the particular patient, disease orcondition to be treated. Examples of other such agents include, interalia, a cytotoxic, anti-cancer or chemotherapeutic agent, ananti-inflammatory or anti-proliferative agent, an antimicrobial orantiviral agent, growth factors, cytokines, an analgesic, atherapeutically active small molecule or polypeptide, a single chainantibody, a classical antibody or fragment thereof, or a nucleic acidmolecule which modulates one or more signaling pathways, and similarmodulating therapeutic molecules which may complement or otherwise bebeneficial in a therapeutic or prophylactic treatment regimen.

Treatment of a patient with a cell-targeting molecule or pharmaceuticalcomposition of the present invention preferably leads to cell death oftargeted cells and/or the inhibition of growth of targeted cells. Assuch, cell-targeting molecules of the present invention, andpharmaceutical compositions comprising them, will be useful in methodsfor treating a variety of pathological disorders in which killing ordepleting target cells may be beneficial, such as, inter alia, cancers,tumors, growth abnormalities, immune disorders, and infected cells. Thepresent invention provides methods for suppressing cell proliferationand treating cell disorders, including neoplasia and/or unwantedproliferation of certain cell-types.

In certain embodiments, the cell-targeting molecules and pharmaceuticalcompositions of the present invention can be used to treat or preventcancers, tumors (malignant and non-malignant), growth abnormalities,immune disorders, and microbial infections. In a further aspect, theabove ex vivo method can be combined with the above in vivo method toprovide methods of treating or preventing rejection in bone marrowtransplant recipients, and for achieving immunological tolerance.

In another aspect, certain embodiments of the cell-targeting moleculesand pharmaceutical compositions of the present invention are endocrineregulating agents—meaning they are capable of treating and/or preventingthe acquisition, development, or consequences of endocrine disordersresulting from endocrine gland hyposecretion, endocrine glandhypersecretion and/or endocrine glandular tumors. In certain furtherembodiments, the cell-targeting molecule of the present inventioncomprises a binding region which is a hormone or hormone analog. Incertain further embodiments, the cell-targeting molecule of the presentinvention is used in a method of reducing endocrine gland hypersecretionby targeting and killing endocrine gland cells. The cell-targetingmolecules and/or pharmaceutical compositions of the present inventionmay be utilized in a method of treating an endocrine disease comprisingthe step of administering to a patient, in need thereof, atherapeutically effective amount of a cell-targeting molecule orpharmaceutical composition of the present invention. In certain furtherembodiments, the disease to be treated is hyperthyroidism and/orhyperparathyroidism

In certain embodiments, the cell-targeting molecules and pharmaceuticalcompositions of the present invention are immunomodulatoryagents—meaning they are capable of treating and/or preventing theacquisition, development, or consequences of immune disorders. Incertain further embodiments, the cell-targeting molecule of the presentinvention comprises a binding region which binds the extracellulartarget biomolecule which is a T-cell receptor (TCR). In certain furtherembodiments, the cell-targeting molecule of the present inventioncomprises a binding region which is a MHC class I tetramer. In certainfurther embodiments, the cell-targeting molecule of the presentinvention is used in a method of reducing the activity and/or viabilityof specific CD8+ cytotoxic T lymphocyte(s) involved in an autoimmunedisorder. The cell-targeting molecules and/or pharmaceuticalcompositions of the present invention may be utilized in a method oftreating an immune disorder comprising the step of administering to apatient, in need thereof a therapeutically effective amount of acell-targeting molecule or pharmaceutical composition of the presentinvention. In certain further embodiments, the disorder to be treated isthe result of tissue destruction by CD8+ T lymphocytes, such as, e.g.,as a result of allograft-related disease.

The cell-targeting molecules and pharmaceutical compositions of thepresent invention are commonly anti-neoplastic agents—meaning they arecapable of treating and/or preventing the development, maturation, orspread of neoplastic or malignant cells by inhibiting the growth and/orcausing the death of cancer or tumor cells. In certain embodiments, thepresent invention provides methods for treating malignancies orneoplasms and other blood cell associated cancers in a mammaliansubject, such as a human, the method comprising the step ofadministering to a subject in need thereof a therapeutically effectiveamount of a cell-targeting molecule or pharmaceutical composition of theinvention.

In another aspect, certain embodiments of the cell-targeting moleculesand pharmaceutical compositions of the present invention areantimicrobial agents—meaning they are capable of treating and/orpreventing the acquisition, development, or consequences ofmicrobiological pathogenic infections, such as caused by viruses,bacteria, fungi, prions, or protozoans.

The cell-targeting molecules and/or pharmaceutical compositions of thepresent invention may be utilized in a method of treating cancercomprising administering to a patient, in need thereof, atherapeutically effective amount of a cell-targeting molecule orpharmaceutical composition of the present invention. In certainembodiments of the methods of the present invention, the cancer beingtreated is selected from the group consisting of: bone cancer (such asmultiple myeloma or Ewing's sarcoma), breast cancer, central/peripheralnervous system cancer (such as brain cancer, neurofibromatosis, orglioblastoma), gastrointestinal cancer (such as stomach cancer orcolorectal cancer), germ cell cancer (such as ovarian cancers andtesticular cancers, glandular cancer (such as pancreatic cancer,parathyroid cancer, pheochromocytoma, salivary gland cancer, or thyroidcancer), head-neck cancer (such as nasopharyngeal cancer, oral cancer,or pharyngeal cancer), hematological cancers (such as leukemia,lymphoma, or myeloma), kidney-urinary tract cancer (such as renal cancerand bladder cancer), liver cancer, lung/pleura cancer (such asmesothelioma, small cell lung carcinoma, or non-small cell lungcarcinoma), prostate cancer, sarcoma (such as angiosarcoma,fibrosarcoma, Kaposi's sarcoma, or synovial sarcoma), skin cancer (suchas basal cell carcinoma, squamous cell carcinoma, or melanoma), anduterine cancer.

The cell-targeting molecules and pharmaceutical compositions of thepresent invention may be utilized in a method of treating an immunedisorder comprising administering to a patient, in need thereof, atherapeutically effective amount of the cell-targeting molecule orpharmaceutical composition of the present invention. In certainembodiments of the methods of the present invention, the immune disorderis related to an inflammation associated with a disease selected fromthe group consisting of: amyloidosis, ankylosing spondylitis, asthma,autism, cardiogenesis, Crohn's disease, diabetes, erythematosus,gastritis, graft rejection, graft-versus-host disease, Grave's disease,Hashimoto's thyroiditis, hemolytic uremic syndrome, HIV-relateddiseases, lupus erythematosus, lymphoproliferative disorders, multiplesclerosis, myasthenia gravis, neuroinflammation, polyarteritis nodosa,polyarthritis, psoriasis, psoriatic arthritis, rheumatoid arthritis,scleroderma, septic shock, Sjögren's syndrome, systemic lupuserythematosus, ulcerative colitis, vasculitis.

Among certain embodiments of the present invention is using thecell-targeting molecule of the present invention as a component of apharmaceutical composition or medicament for the treatment or preventionof a cancer, tumor, other growth abnormality, immune disorder, and/ormicrobial infection. For example, immune disorders presenting on theskin of a patient may be treated with such a medicament in efforts toreduce inflammation. In another example, skin tumors may be treated withsuch a medicament in efforts to reduce tumor size or eliminate the tumorcompletely.

Among certain embodiments of the present invention is a method of usinga cell-targeting molecule, pharmaceutical composition, and/or diagnosticcomposition of the present invention for the purpose of informationgathering regarding diseases, conditions and/or disorders. For example,the cell-targeting molecule of the present invention may be used forimaging of pMHC I presentation by tumor cells using antibodies specificto certain pMHC Is. The detection of such labeled target cells afterbeing treated with a cell-targeting molecule of the present inventionmay provide a readout regarding a targeted cell-type's competency atantigen processing and MHC class I presentation as well as thepercentage of such competent target cells within a population of targetcells when combined with readouts from diagnostic variants of thecell-targeting molecules of the invention.

Among certain embodiments of the present invention is a method of usinga cell-targeting molecule, pharmaceutical composition, and/or diagnosticcomposition of the present invention to detect the presence of acell-type for the purpose of information gathering regarding diseases,conditions and/or disorders. The method comprises contacting a cell witha diagnostically sufficient amount of a cell-targeting molecule of thepresent invention in order to detect the molecule by an assay ordiagnostic technique. The phrase “diagnostically sufficient amount”refers to an amount that provides adequate detection and accuratemeasurement for information gathering purposes by the particular assayor diagnostic technique utilized. Generally, the diagnosticallysufficient amount for whole organism in vivo diagnostic use will be anon-cumulative dose of between 0.01 mg to 10 mg of the detectionpromoting agent linked cell-targeting molecule of the invention per kgof subject per subject. Typically, the amount of cell-targeting moleculeof the invention used in these information-gathering methods will be aslow as possible provided that it is still a diagnostically sufficientamount. For example, for in vivo detection in an organism, the amount ofcell-targeting molecule or diagnostic composition of the inventionadministered to a subject will be as low as feasibly possible.

The cell-type specific targeting of cell-targeting molecules of thepresent invention combined with detection promoting agents provides away to detect and image cells physically coupled with an extracellulartarget biomolecule of a binding region of the molecule of the invention.Alternatively, the display of a cell-targeting molecule deliveredheterologous, CD8+ T-cell epitope cargo can provide a way to detect andimage cells which internalized a cell-targeting molecule of the presentinvention. Imaging of cells using the cell-targeting molecules anddiagnostic compositions of the present invention may be performed invitro or in vivo by any suitable technique known in the art. Diagnosticinformation may be collected using various methods known in the art,including whole body imaging of an organism or using ex vivo samplestaken from an organism. The term “sample” used herein refers to anynumber of things, but not limited to, fluids such as blood, urine,serum, lymph, saliva, anal secretions, vaginal secretions, and semen,and tissues obtained by biopsy procedures. For example, variousdetection promoting agents may be utilized for non-invasive in vivotumor imaging by techniques such as magnetic resonance imaging (MRI),optical methods (such as direct, fluorescent, and bioluminescentimaging), positron emission tomography (PET), single-photon emissioncomputed tomography (SPECT), ultrasound, x-ray computed tomography, andcombinations of the aforementioned (see, Kaur S et al., Cancer Lett 315:97-111 (2012), for review).

Among certain embodiment of the present invention is a method of using acell-targeting molecule, pharmaceutical composition, and/or diagnosticcomposition of the present invention to label or detect the interiors ofneoplastic cells and/or immune cell-types (see e.g., Koyama Y et al.,Clin Cancer Res 13: 2936-45 (2007); Ogawa M et al., Cancer Res 69:1268-72 (2009); Yang L et al., Small 5: 235-43 (2009)). This may bebased on the ability of certain cell-targeting molecules of the presentinvention to enter specific cell-types and route within cells viaretrograde intracellular transport to specific subcellular compartmentssuch that interior compartments of specific cell-types are labeled fordetection. This can be performed on cells in situ within a patient or invitro on cells and tissues removed from an organism, e.g. biopsymaterials.

Diagnostic compositions of the present invention may be used tocharacterize a disease, disorder, or condition as potentially treatableby a related pharmaceutical composition of the present invention.Certain compositions of matter of the present invention may be used todetermine whether a patient belongs to a group that responds to atherapeutic strategy which makes use of a cell-targeting molecule of theinvention, or composition thereof, or related method of the presentinvention as described herein or is well suited for using a deliverydevice of the invention.

Diagnostic compositions of the present invention may be used after adisease, e.g. a cancer, is detected in order to better characterize it,such as to monitor distant metastases, heterogeneity, and stage ofcancer progression. The phenotypic assessment of disease disorder orinfection can help prognostic and prediction during therapeutic decisionmaking. In disease reoccurrence, certain methods of the invention may beused to determine if a localized or systemic problem.

Diagnostic compositions of the present invention may be used to assessresponses to therapeutic(s) regardless of the type of therapeutic, e.g.small molecule drug, biological drug, or cell-based therapy. Forexample, certain embodiments of the diagnostic compositions of theinvention may be used to measure changes in tumor size, changes inantigen positive cell populations including number and distribution, ormonitoring a different marker than the antigen targeted by a therapyalready being administered to a patient (see Smith-Jones P et al., Nat.Biotechnol 22: 701-6 (2004); Evans M et al., Proc. Natl. Acad. Sci.U.S.A. 108: 9578-82 (2011)).

Diagnostic compositions of the present invention may be used to assessthe MHC class I system functionality in target cell-types. For example,certain malignant cells, such as infected, tumor, or cancer cells, canexhibit alterations, defects, and perturbations to their MHC class Ipresentation pathways. This can be studied in vitro or in vivo.Diagnostic compositions of the invention may be used to monitor changesin MHC class I presentation among individual cells within a populationof target cells within an organism or to count or determine percentagesof MHC class I presentation defective target cells within an organism,tumor biopsy, etc.

In certain embodiments of the method used to detect the presence of acell-type may be used to gather information regarding diseases,disorders, and conditions, such as, for example bone cancer (such asmultiple myeloma or Ewing's sarcoma), breast cancer, central/peripheralnervous system cancer (such as brain cancer, neurofibromatosis, orglioblastoma), gastrointestinal cancer (such as stomach cancer orcolorectal cancer), germ cell cancer (such as ovarian cancers andtesticular cancers, glandular cancer (such as pancreatic cancer,parathyroid cancer, pheochromocytoma, salivary gland cancer, or thyroidcancer), head-neck cancer (such as nasopharyngeal cancer, oral cancer,or pharyngeal cancer), hematological cancers (such as leukemia,lymphoma, or myeloma), kidney-urinary tract cancer (such as renal cancerand bladder cancer), liver cancer, lung/pleura cancer (such asmesothelioma, small cell lung carcinoma, or non-small cell lungcarcinoma), prostate cancer, sarcoma (such as angiosarcoma,fibrosarcoma, Kaposi's sarcoma, or synovial sarcoma), skin cancer (suchas basal cell carcinoma, squamous cell carcinoma, or melanoma), uterinecancer, AIDS, amyloidosis, ankylosing spondylitis, asthma, autism,cardiogenesis, Crohn's disease, diabetes, erythematosus, gastritis,graft rejection, graft-versus-host disease, Grave's disease, Hashimoto'sthyroiditis, hemolytic uremic syndrome. HIV-related diseases, lupuserythematosus, lymphoproliferative disorders, multiple sclerosis,myasthenia gravis, neuroinflammation, polyarteritis nodosa,polyarthritis, psoriasis, psoriatic arthritis, rheumatoid arthritis,scleroderma, septic shock, Sjögren's syndrome, systemic lupuserythematosus, ulcerative colitis, vasculitis, cell proliferation,inflammation, leukocyte activation, leukocyte adhesion, leukocytechemotaxis, leukocyte maturation, leukocyte migration, neuronaldifferentiation, acute lymphoblastic leukemia (ALL), T acute lymphocyticleukemia/lymphoma (ALL), acute myelogenous leukemia, acute myeloidleukemia (AML), B-cell chronic lymphocytic leukemia (B-CLL), B-cellprolymphocytic lymphoma, Burkitt's lymphoma (BL), chronic lymphocyticleukemia (CLL), chronic myelogenous leukemia (CML-BP), chronic myeloidleukemia (CML), diffuse large B-cell lymphoma, follicular lymphoma,hairy cell leukemia (HCL), Hodgkin's Lymphoma (HL), intravascular largeB-cell lymphoma, lymphomatoid granulomatosis, lymphoplasmacyticlymphoma, MALT lymphoma, mantle cell lymphoma, multiple myeloma (MM),natural killer cell leukemia, nodal marginal B-cell lymphoma,Non-Hodgkin's lymphoma (NHL), plasma cell leukemia, plasmacytoma,primary effusion lymphoma, pro-lymphocytic leukemia, promyelocyticleukemia, small lymphocytic lymphoma, splenic marginal zone lymphoma,T-cell lymphoma (TCL), heavy chain disease, monoclonal gammopathy,monoclonal immunoglobulin deposition disease, myelodusplastic syndromes(MDS), smoldering multiple myeloma, and Waldenström macroglobulinemia.

In certain embodiments, the cell-targeting molecules of the presentinvention, or pharmaceutical compositions thereof are used for bothdiagnosis and treatment, or for diagnosis alone. In some situations, itwould be desirable to determine or verify the HLA variant(s) and/or HLAalleles expressed in the subject and/or diseased tissue from thesubject, such as, e.g., a patient in need of treatment, before selectinga cell-targeting molecule of the invention for use in treatment(s). Insome situations, it would be desirable to determine, for an individualsubject, the immunogenicity of certain CD8+ T-cell epitopes beforeselecting which cell-targeting molecule, or composition thereof, to usein a method of the present invention.

The present invention is further illustrated by the followingnon-limiting examples of cell-targeting molecules comprising theaforementioned structures and functions, in particular the function ofextracellular targeting the delivery of a CD8+ T-cell epitope cargo tospecific cells and then intracellular delivery of the CD8+ T-cellepitope cargo to the MHC class I pathway for presentation of thedelivered CD8+ T-cell epitope cargo complexed with MHC class I moleculeson a cell surface.

EXAMPLES

De-immunized, Shiga toxin effector polypeptides can be engineered todeliver immunogenic epitope-peptides for presentation by cells in whichthese polypeptides are present. Furthermore, de-immunized, Shiga toxineffector polypeptides that are furin-cleavage resistant can also beengineered to deliver immunogenic epitope-peptides for presentation bytarget cells. Cell-targeting molecules comprising such de-immunized,Shiga toxin effector polypeptides provide for the targeted delivery ofepitopes to specific cells and may be used in applications involvingcell-type specific presentation of immuno-stimulatory epitopes within achordate. The presentation of a T-cell immunogenic epitope by the MHCclass I system within a chordate targets the epitope presenting cell forkilling by CD8+ CTL-mediated lysis and may also stimulate other immuneresponses in the vicinity.

In the Examples, CD8+ T-cell antigens were fused to cell-targetingmolecules comprising de-immunized and furin-cleavage resistant Shigatoxin A Subunit effector polypeptides. All these fusion polypeptidesinvolve the addition of at least one peptide to the starting polypeptidescaffold and do not require the embedding or inserting of anyheterologous, CD8+ T-cell epitope internally within a Shiga toxineffector polypeptide component, although other embedded or insertedheterologous, CD8+ T-cell epitope may be present in the deimmunized,Shiga toxin effector polypeptide. Thus, in certain exemplarycell-targeting molecules of the present invention, the Shiga toxineffector polypeptide consists of a de-immunized Shiga toxin polypeptidethat may further be furin-cleavage resistant and/or comprise one or moreembedded or inserted CD8+ T-cell epitopes.

The Examples below describe exemplary, cell-targeting molecules of thepresent invention comprising (1) an immunoglobulin-type binding regionfor cell-targeting, (2) a de-immunized and furin-cleavage resistantShiga toxin effector polypeptide, and (3) a cargo consisting of a fused,heterologous, CD8+ T-cell epitope-peptide which is neither embedded norinserted into a Shiga toxin effector polypeptide region. Theseexemplary, cell-targeting molecules of the present invention bind totarget biomolecules expressed by targeted cell-types and enter targetedcells. Then, the internalized exemplary cell-targeting moleculeseffectively route their Shiga toxin effector polypeptide components tothe cytosol and optionally kill target cells directly via ribosomeinhibition.

The Examples below demonstrate that an exemplary cell-targeting moleculedelivered, within target cells, its fused, heterologous, CD8+ T-cellepitope-peptide cargo to the MHC class I pathway resulting inpresentation of the T-cell epitope-peptide on the surface of targetcells. The cell-surface display of delivered T-cell epitopes complex toMHC class I molecules by a target cell can signal to CD8+ effectorT-cells to kill the epitope-displaying target cells as well as stimulateother immune responses in the vicinity of the epitope-displaying targetcell.

As demonstrated below in Example 1, a cell-targeting molecule of thepresent invention was capable, upon exogenous administration, ofdelivering a heterologous, T-cell epitope-peptide to the MHC class Ipathway for presentation by targeted, human, cancer cells. Alsodemonstrated below in Examples 1-2, two cell-targeting molecules of thepresent invention were capable of specifically killing target-expressinghuman, cancer cells via their de-immunized and furin-cleavage resistantShiga toxin effector polypeptide components. Further demonstrated belowin Example 2, two other cell-targeting molecules of the presentinvention were capable of killing target-expressing human, cancer cellsvia the action of immune cells in human PBMC coculture experiments. Inaddition, Example 2 demonstrates that a catalytically activecell-targeting molecule of the present invention can kill moretarget-expressing human cancer cells cocultured with human PBMCs than acatalytically active reference molecule lacking any heterologous CD8+T-cell epitope-cargo-suggesting that inducing both direct cell-kill andindirect intercellular T-cell killing mechanisms can achieve greatertarget cell-killing in the presence of the appropriate MHC class Iepitope-specific restricted T-cells.

Example 1. Cell-Targeting Molecules Comprising De-Immunized andFurin-Cleavage Resistant, Shiga Toxin A Subunit Derived Polypeptides andFused, T-Cell Epitope-Peptides

Cell-targeting molecules are created and tested—the cell-targetingmolecules each comprising 1) a cell-targeting binding region, 2) ade-immunized Shiga toxin effector polypeptide which is optionallyfurin-cleavage resistant, and 3) at least one T-cell epitope-peptidecargo consisting of a fused, heterologous, CD8+ T-cell epitope-peptidewhich is neither embedded nor inserted into a Shiga toxin effectorpolypeptide region. Previously, Shiga toxin A Subunit derived,cell-targeting molecules have been constructed and shown to promotecellular internalization and direct intracellular routing of their Shigatoxin effector polypeptide components to the cytosol (see e.g. WO2014/164680, WO 2014/164693, WO 2015/138435, WO 2015/138452, WO2015/113005, WO 2015/113007, WO 2015/191764, WO 2016/196344, and WO2018/106895). T-cell epitope-peptides are fused to modular polypeptidecomponents of these de-immunized and/or furin-cleavage resistant Shigatoxin A Subunit derived, cell-targeting molecules in order to createnovel cell-targeting molecules each having at least one T-cellepitope-peptide that is neither embedded nor inserted into a Shiga toxinA1 fragment derived component and which is heterologous to the Shigatoxin A subunit component (see e.g. WO 2017/019623).

As demonstrated below in this Example, a cell-targeting protein of thepresent invention was capable, upon exogenous administration, ofdelivering a heterologous, T-cell epitope-peptide to the MHC class Ipathway for presentation by targeted, human, cancer cells. Alsodemonstrated below in this Example, a cell-targeting protein of thepresent invention was capable of specifically killing target-expressinghuman, cancer cells via its de-immunized and furin-cleavage resistantShiga toxin effector polypeptide component. The cell-targeting bindingregion of the exemplary cell-targeting protein of this Example wascapable of exhibiting high-affinity binding to an extracellular targetbiomolecule physically-coupled to the surface of a specificcell-type(s). The exemplary cell-targeting protein of this Example iscapable of selectively targeting cells expressing a target biomoleculeof their cell-targeting binding region and internalizing into thesetarget cells.

1. Construction of Exemplary Cell-Targeting Molecules of the PresentInvention

Using techniques known in the art, exemplary cell-targeting fusionproteins are created by genetically fusing a human CD8+ T-cellepitope-peptide to the amino terminus (N-terminus) or carboxy terminus(C-terminus) of a polypeptide component of a parental, cell-targetingprotein comprising 1) a de-immunized and furin-cleavage resistant Shigatoxin A Subunit effector polypeptide and 2) a cell-targeting bindingregion polypeptide separated by a proteinaceous linker. The fused, CD8+T-cell epitope cargos are chosen from among several T-cellepitope-peptides originating in viruses that commonly infect humans. Theresulting cell-targeting, fusion proteins are constructed such that eachcomprised a single, continuous polypeptide comprising a cell-targeting,binding region polypeptide, a de-immunized and furin-cleavage resistantShiga toxin A Subunit effector polypeptide, and a fused, heterologous,CD8+ T-cell epitope cargo.

A cell-targeting molecule of the present invention may comprise (1) animmunoglobulin-type binding region for cell-targeting, (2) ade-immunized and furin-cleavage resistant Shiga toxin effectorpolypeptide, and (3) a cargo consisting of a fused, heterologous, CD8+T-cell epitope-peptide which is neither embedded nor inserted into aShiga toxin effector polypeptide region. All three components may bechosen from the prior art or created using routine methods known to theskilled worker (see e.g. WO 2014/164680. WO 2014/164693, WO 2015/138435,WO 2015/138452, WO 2015/113005, WO 2015/113007, WO 2015/191764, WO2016/196344, and WO 2018/106895).

Immunoglobulin-type binding regions have been described previously in WO2014/164680, WO 2014/164693. WO 2015/138435, WO 2015/138452, WO2015/113005, WO 2015/113007, WO 2015/191764, and WO 2016/196344.

De-immunized and furin-cleavage resistant, Shiga toxin effectorpolypeptides have been described previously in WO 2015/113007, WO2015/191764, and WO 2016/196344.

Heterologous, CD8+ T-cell epitope-peptides have been describedpreviously in WO 2015/113005 and WO 2016/196344.

In this Example, proteinaceous linkers are selected from the prior artto link the components.

All the Shiga toxin effector polypeptide components of thecell-targeting molecules of this Example are derived from amino acids1-251 of SLT-1A (SEQ ID NO: 1), and some of them contained two or moreamino acid residue substitutions relative to a wild-type Shiga toxin ASubunit, such as, e.g., de-immunizing substitutions and/or substitutionsfurin-cleavage motif disrupting mutations (see e.g. WO 2015/113007, WO2015/191764, and WO 2016/196344). Exemplary, Shiga toxin effectorpolypeptide components of the cell-targeting molecules of the inventionare SEQ ID NOs: 29-38.

All of the cell-targeting molecules tested in the experiments of thisExample were produced in a bacterial system and purified by columnchromatography using techniques known to the skilled worker.

The exemplary cell-targeting molecule of the present invention that wasproduced and tested in this Example was SLT-1A-DI-FR::scFv1::C2 (SEQ IDNO:252). This exemplary, cell-targeting, fusion protein of the presentinvention comprised a cell-targeting binding region comprising asingle-chain variable fragment component (scFv1), a de-immunized andfurin-cleavage resistant Shiga toxin A Subunit effector polypeptidecomponent (SLT-1A-DI-FR), and a human CD8+ T-cell epitope-peptide (C2)fused to the binding region. The immunoglobulin-type binding regionscFv1 is a single-chain variable fragment which bound with high-affinityto a certain cell-surface, target biomolecule physically coupled to thesurface of certain human cancer cells. The Shiga toxin effectorpolypeptide component of the cell-targeting molecule of this Example wasSLT-1A-DI-FR (SEQ ID NO:29). The epitope-peptide C2 (SEQ ID NO:21)selected as a fused cargo was known to be immunogenic. Thecell-targeting molecule SLT-1A-DI-FR::scFv1::C2 (SEQ ID NO:252) wasproduced in a bacterial system and purified by column chromatographyusing techniques known to the skilled worker.

After purification, the multimeric state of the cell-targeting moleculeSLT-1A-DI-FR::scFv1::C2 (SEQ ID NO:252) was tested using bothsize-exclusion chromatography (SEC) and sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) under denaturingconditions.

A sample of SLT-1A-DI-FR::scFv1 protein (SEQ ID NO:258) was analyzed bySEC using a Superdex 200 30/3(00) column (GE Healthcare, LittleChalfont, Buckinghamshire, U.K.) having a 24-mL bed volume. The samplewas loaded onto the column and at least 24 mL of buffer was flowed overthe column while an ultraviolet light (UV) detector monitored theelution of protein from by absorbance at 280 nm reported asmilli-absorbance units (mAU). Molecules with smaller molecular weightsare retarded when flowing through matrixes used for size exclusionchromatography as compared to larger molecules and therefore smallmolecules exhibit longer size exclusion chromatography retention timesthan larger molecules. Using the elution rates of proteins of knownmolecular masses subjected to size exclusion chromatography with thesame column and conditions as references, the molecular mass of theSLT-1A-DI-FR::scFv I protein (SEQ ID NO:258) sample under nativeconditions was estimated.

The SLT-1A-DI-FR::scFv1 (SEQ ID NO:258) sample analyzed produced aprimary peak corresponding to a retention of 13.2 mL, which based on thereference proteins of known masses corresponds to a molecular mass ofabout 110 kiloDalton (kDa) (FIG. 4), which was consistent with a 110 kDahomodimeric protein comprising two polypeptides each having a mass of 55kDa. By this SEC analysis, a sample of SLT-1A-DI-FR::scFv1::C2 protein(SEQ ID NO:252) is measured to be about the same size, which appearscongruent with an approximately 110 kDa molecule consisting of twopolypeptides each having a mass of 55 kDa (see below).

Protein samples of SLT-1A-DI-FR::scFv1::C2 (SEQ ID NO:252) andSLT-1A-DI-FR::scFv1 (SEQ ID NO:258) were loaded in equal amounts onto4-20% sodium dodecyl sulfate (SDS) polyacrylamide gels (Lonza, Basel,CH) and electrophoresed under denaturing conditions (FIG. 5). Theresulting gels were analyzed by Coomassie staining. A molecular weight(MW) marker (ProSeive™ QuadColor™, Lonza, Basel, CH) was loaded toindicate approximate molecular weight of the proteins loaded on the gel.Under these denaturing conditions, any multimeric protein complexes wereexpected to dissociate into monomeric polypeptides. BothSLT-1A-DI-FR::scFv1:C2 and SLT-1A-DI-FR::scFv1 samples formed bands withapparent molecular weights of about 55 kDa (FIG. 5), which correspondsto the approximate molecular weight of the protein mass predicted foreither SLT-1A-DI-FR::scFv1::C2 (SEQ ID NO:252) or SLT-1A-DI-FR::scFv1(SEQ ID NO:258) for having 508 and 500 amino acids, respectively.

II. Testing the Shiga Toxin A Subunit Effector Polypeptide Components ofCell-Targeting Molecules for Retention of Shiga Toxin Functions afterthe Fusion of Binding Regions and T-Cell Epitope-Peptides

Exemplary cell-targeting proteins are tested for retention of Shigatoxin A Subunit effector functions after the fusion of heterologous,CD8+ T-cell epitope-peptides. The Shiga toxin A Subunit effectorfunctions analyzed were cytotoxicity, and by inference self-directingsubcellular routing to the cytosol.

Testing the Cytotoxic Activities of Exemplary Cell-Targeting Moleculesof the Invention

The cytotoxic activities of exemplary cell-targeting molecules of theinvention are measured using a tissue culture cell-based toxicity assay.The concentration of exogenously administered cell-targeting moleculewhich kills half the cells in a homogenous cell population (half-maximalcytotoxic concentration) was determined for certain cell-targetingmolecules of the invention. The cytotoxicities of exemplarycell-targeting molecules are tested using cell-kill assays involvingeither target biomolecule positive or target biomolecule negative cellswith respect to the target biomolecule of each cell-targeting molecule'sbinding region.

The target cells used in this Example (cell lines A. B, and C) wereimmortalized human cancer cells available from the ATCC (Manassas Va.,U.S.) or the DSMZ (The Leibniz Deutsche Sammlung von Mikroorganismen undZellkulture) (Braunschweig, Del.)).

The cell-kill assays were performed as follows. Human tumor cell linecells were plated (typically at 2×10³ cells per well for adherent cells,plated the day prior to protein addition, or 7.5×10³ cells per well forsuspension cells, plated the same day as protein addition) in 20 μL cellculture medium in 384-well plates. A series of 10-fold dilutions of theproteins to be tested was prepared in an appropriate buffer, and 5 μL ofthe dilutions or only buffer as a negative control were added to thecells. Control wells containing only cell culture medium were used forbaseline correction. The cell samples were incubated with the proteinsor just buffer for 3 or 5 days at 37° C. and in an atmosphere of 5%carbon dioxide (CO₂). The total cell survival or percent viability wasdetermined using a luminescent readout using the CellTiter-Glo,Luminescent Cell Viability Assay (G7573 Promega Madison, W1, U.S.)according to the manufacturer's instructions as measured in relativelight units (RLU).

The Percent Viability of experimental wells was calculated using thefollowing equation: (Test RLU−Average Media RLU)+(Average CellsRLU−Average Media RLU)×100. Log protein concentration versus PercentViability was plotted in Prism (GraphPad Prism, San Diego, Calif., U.S.)and log (inhibitor) versus response (3 parameter) analysis were used todetermine the half-maximal cytotoxic concentration (CD₅₀) value for thetested proteins. The CD₅₀ values for each exemplary cell-targetingprotein tested was calculated when possible.

The specificity of the cytotoxic activity of a given cell-targetingmolecule was determined by comparing cell kill activities toward cellsexpressing a significant amount of a target biomolecule of the bindingregion of the cell-targeting molecule (target positive cells) withcell-kill activities toward cells which do not exhibit any significantamount of any target biomolecule of the binding region of thecell-targeting molecule physically coupled to any cellular surface(target negative cells). This was accomplished by determining thehalf-maximal cytotoxic concentrations of a given cell-targeting moleculeof the invention toward cell populations which were positive for cellsurface expression of the target biomolecule of the cell-targetingmolecule being analyzed, and, then, using the same cell-targetingmolecule concentration range to attempt to determine the half-maximalcytotoxic concentrations toward cell populations which were negative forcell surface expression of the target biomolecule of the cell-targetingmolecule. In some experiments, the target negative cells treated withthe maximum amount of the Shia-toxin containing molecule did not showany change in viability as compared to a “buffer only” negative control.

The cytotoxic activity levels of various molecules tested using thecell-kill assay described above are reported in Table 1. As reported inTable 1, exemplary cell targeting proteins of the invention which weretested in this assay exhibited potent cytotoxicity. While the fusion ofa heterologous, CD8+ T-cell epitope-peptide to a Shiga toxin derived,cell-targeting protein can result in no change in cytotoxicit, someexemplary cell-targeting proteins exhibited reduced cytotoxicity ascompared to the parental protein from which it was derived, which didnot comprise any fused, heterologous epitope-peptide (Table I). Asreported in the Examples, a molecule exhibiting a CD₅₀ value within10-fold of a CD₅₀ value measured for a reference molecule is consideredto exhibit cytotoxic activity comparable to that reference molecule. Inparticular, any exemplary cell-targeting molecule of the presentinvention that exhibited a CD₅₀ value to a target positive cellpopulation within 10-fold of the CD₅₀ value of a referencecell-targeting molecule comprising the same binding region and awild-type, Shiga toxin effector polypeptide (e.g. SLT-1A-WT (SEQ IDNO:279)) but not comprising any fused, heterologous, T-cellepitope-peptide, toward the same cell-type is referred to herein as“comparable to wild-type.” Cell-targeting molecules that exhibited aCD₅₀ value to a target positive cell population within 100-fold to10-fold of a reference molecule comprising the same binding region andthe same Shiga toxin effector polypeptide but not comprising any fused,heterologous, T-cell epitope-peptide is referred to herein as active but“attenuated.”

TABLE 1 Cytotoxic Activities of Shiga Toxin Derived, Cell-TargetingProteins Comprising Fused, Heterologous Epitope-Peptides AdministeredFused Fusion Cell Line A Cell Line B Cell Line C Molecule EpitopeLocation (target positive) (target positive) (target positive)SLT-1A-DI- C2 carboxy- 0.22 0.91 0.86 FR::scFv1::C2 terminus SLT-1A-DI-none N/A 0.022 0.21 0.18 FR::scFv1

Table 1 and FIG. 2 show that SLT-1A-DI-FR::scFv1::C2 (SEQ ID NO:252)exhibits a cytotoxicity to three different types of target positivecells with a similar cytotoxic activity to a parental protein lackingthe fused antigen C2 (SEQ ID NO:258).

III. Testing Epitope-Peptide Delivery and Cell-Surface Presentation ofDelivered Epitope-Peptides by Target Cells

The successful delivery of a T-cell epitope can be determined bydetecting specific cell surface, MHC class I molecule/epitope complexes(pMHC Is). In order to test whether a cell-targeting protein can delivera fused T-cell epitope to the MHC class I presentation pathway of targetcells, an assay was employed which detects human, MHC Class I moleculescomplexed with specific epitopes. A flow cytometry method was used todemonstrate delivery of a T-cell epitope (fused to a de-immunized andfurin-cleavage resistant, Shiga toxin A Subunit derived cell-targetingprotein) and extracellular display of the delivered T-cellepitope-peptide in complex with MHC Class I molecules on the surfaces oftarget cells. This flow cytometry method utilizes soluble human T-cellreceptor (TCR) multimer reagents (Soluble T-Cell Antigen Receptor STAR™Multimer, Altor Bioscience Corp., Miramar, Fla., U.S.), each withhigh-affinity binding to a different epitope-human HLA complex.

Each STAR™ TCR multimer reagent is derived from a specific T-cellreceptor and allows detection of a specific peptide-MHC complex based onthe ability of the chosen TCR to recognize a specific peptide presentedin the context of a particular MHC class I molecule. These TCR multimersare composed of recombinant human TCRs which have been biotinylated andmultimerized with streptavidin. The TCR multimers are labeled withphycoerythrin (PE). These TCR multimer reagents allow the detection ofspecific peptide-MHC Class I complexes presented on the surfaces ofhuman cells because each soluble TCR multimer type recognizes and stablybinds to a specific peptide-MHC complex under varied conditions (Zhu Xet al., J Immunol 176: 3223-32 (2006)). These TCR multimer reagentsallow the identification and quantitation by flow cytometry ofpeptide-MHC class I complexes present on the surfaces of cells.

The TCR CMV-pp65-PE STAR™ multimer reagent (Altor Bioscience Corp.,Miramar, Fla., U.S.) was used in this Example. MHC class I pathwaypresentation of the human CMV C2 peptide (NLVPMVATV (SEQ ID NO:21)) byhuman cells expressing the HLA-A2 can be detected with the TCRCMV-pp65-PE STAR™ multimer reagent which exhibits high affinityrecognition of the CMV-pp65 epitope-peptide (residues 495-503, NLVPMVATV(SEQ ID NO:21)) complexed to human HLA-A2 and is labeled with PE.

Using standard flow cytometry methods known in the art, the target cellswere confirmed to express on their cell surfaces both the HLA-A2MHC-Class I molecule and the extracellular target biomolecules of thecell-targeting proteins used in this Example. In some experiments, thehuman cancer cells were pretreated with human interferon gamma (IFN-γ)to enhance expression of human HLA-A2.

Sets of target cells were treated by exogenous administration ofSLT-1A-DI-FR::scFv1::C2 (SEQ ID NO:252), a cell-targeting moleculecomprising a carboxy-terminal fused, viral, CD8+ T-cell epitope, or weretreated by exogenous administration of a negative-control cell-targetingfusion protein which did not comprise any fused, heterologous, viralepitope-peptide, SLTA-1A-DI-FR::scFv1 (SEQ ID NO:258). Thecell-targeting molecules and reference molecules used in theseexperiments were both catalytically active, cytotoxic cell-targetingmolecules. These treatments were at cell-targeting moleculeconcentrations similar to those used by others taking into accountcell-type specific sensitivities to Shiga toxins (see e.g. WO2015/113005). The treated cells were then incubated for 4-16 hours instandard conditions, including at 37° C., and an atmosphere with 5%carbon dioxide, to allow for intoxication mediated by a de-immunized andfurin-cleavage resistant, Shiga toxin effector polypeptide. Then thecells were washed and incubated with the TCR CMV-pp65-PE STAR™ multimerreagent to “stain” C2 peptide-HLA-A2 complex-presenting cells.

As controls, sets of target cells are treated in three conditions: 1)without any treatment (“untreated”) meaning there was addition of onlybuffer to the cells and no addition of any exogenous molecules, 2) withexogenously administered CMV C2 peptide (CMV-pp65, aa495-503: sequenceNLVPMVATV (SEQ ID NO:21), synthesized by BioSynthesis, Lewisville, Tex.,U.S.), and/or 3) with exogenously administered CMV C2 peptide ((SEQ IDNO:21), as above) combined with a Peptide Loading Enhancer (“PLE,” AltorBioscience Corp., Miramar, Fla. U.S.). The C2 peptide (SEQ ID NO:21)combined with PLE treatment allowed for exogenous peptide loading andserved as a positive control. Cells displaying the appropriate MHC classI haplotype can be forced to load the appropriate exogenously appliedpeptide from an extracellular space (i.e. in the absence of cellularinternalization of the applied peptide) or in the presence of PLE, whichis a mixture of B2-microglobulin and other components.

After the treatments, all the sets of cells were washed and incubatedwith the TCR CMV-pp65-PE STAR™ multimer reagent for one hour on ice. Thecells were washed and the fluorescence of the samples was measured byflow cytometry using an Accuri™ C6 flow cytometer (BD Biosciences, SanJose, Calif., U.S.) to detect the presence of and quantify any TCRCMV-pp65-PE STAR™ multimer bound to cells in the population (sometimesreferred to herein as “staining”) in relative light units (RLU).

Table 2 and FIGS. 4-8 show results from experiments using the TCR STAR™assay detecting cell-surface complexes of C2 epitope/HLA-A2 MHC class Imolecule. For each experiment, the untreated control sample was used toidentify the positive and negative cell populations by employing a gatewhich results in less than 1% of cells from the untreated control in the“positive” gate (representing background signal). The same gate was thenapplied to the other samples to characterize the positive population foreach sample. Positive cells in this assay were cells which were bound bythe TCR-CMV-pp65-PE STAR™ reagent and counted in the positive gatedescribed above.

In FIG. 3, the flow cytometry histogram is given with the cell counts(number of cells or simply “counts”) on the Y-axis and the relativefluorescent units (RFU) representing TCR CMV-pp65 STAR™ multimer, PEstaining signal on the X-axis (log scale). The black line shows theresults for the untreated-cells-only sample, and the gray line shows theresults for the negative controls (treatment with only a parentalcell-targeting protein lacking any viral epitope-peptide), or thetreatment with a specific, exemplary, cell-targeting protein of theinvention. In FIG. 3, the top panel shows the results for the untreatedcell samples using a black line and the results for the cell-targetingmolecule with a fused antigen, SLTA-1A-DI-FR::scFv1::C2, treated samplesusing a gray line. In FIG. 3, the middle panel shows the results foruntreated cell samples using a black line and the results for thecontrol protein, SLTA-1A-DI-FR::scFv1, which did not comprise any fusedepitope-peptide, using a gray line. In FIG. 3, the bottom panel showsthe results the results for the control protein, SLTA-1A-DI-FR::scFv1,which did not comprise any fused epitope-peptide, using a black line andthe results for the cell-targeting molecule with the fused epitope,SLTA-1A-DI-FR::scFv1::C2, treated samples using a gray line.

TABLE 2 Detection of Cell Surface, MHC Class I/C2 Epitope Complexesafter Delivery of C2 Epitope-Peptides by Cell-Targeting Proteins:Peptide-epitope C2/MHC class I complexes detected on the sigfaces ofintoxicated, target cells incubation percentage of target positiveduration pMHC I complex iMFI Protein cell-type (hours) presenting cells(RFU) Experiment 1 SLT-1A-DI-FR::scFv1::C2 Cell Line B 16 hours 35.0%10,090 SLT-1A-DI-FR::scFv2 Cell Line B 16 hours  4.8% 964 No proteinCell Line B 16 hours   0% 14

As seen in Table 2 and FIG. 3, cell samples treated with an exemplarycell-targeting protein of the present invention (SEQ ID NO:252)displayed expression of the C2-epitope/HLA-A2 MHC class I moleculecomplex on the surfaces of the treated cells. In contrast, cells thatwere treated with the parental cell-targeting protein SLT-1A-DI-FR::scFv(SEQ ID NO:258), which did not contain any fused T-cell epitope-peptideas a negative control, exhibited positive cell staining of less thanfive percent of the cells in the treated cell population (Table 2: FIG.3). Due to processing efficiency and kinetics, which were not measured,it is possible that the percentage of presented C2-epitope/HLA-A2complexes detected at a single time-point in a “cell-targeting protein”treatment sample may not accurately reflect the maximum quantity ofC2-epitope/HLA-A2 presentation possible after delivery by a given,exemplary, cell-targeting protein of the present invention.

The detection of the T-cell epitope C2 (SEQ ID NO:21) complexed withhuman MHC Class I molecules (C2 epitope-peptide/HLA-A2) on the cellsurface of cell-targeting molecule treated target cells demonstratedthat exemplary cell-targeting protein SLTA-1A-DI-FR::scFv1::C2 (SEQ IDNO:252) comprising this fused epitope-peptide C2 (SEQ ID NO:21) and ade-immunized, furin-cleavage resistant, Shiga toxin effectorpolypeptide, were capable of entering target cells, performingsufficient sub-cellular routing, and delivering sufficient C2 (SEQ IDNO:21) epitope to the MHC class I pathway for surface presentation bytarget cells.

IV. Testing Cytotoxic T-Cell Mediated Cytolysis of Intoxicated TargetCells and Other Immune Responses Triggered by MHC Class I Presentationof T-Cell Epitopes Delivered by Cell-Targeting Molecules of the PresentInvention

In this Example, standard assays known in the art are used toinvestigate the functional consequences of target cells' MHC class Ipresentation of T-cell epitopes delivered by exemplary cell-targetingmolecules of the invention. The functional consequences to investigateinclude CTL activation (e.g. signal cascade induction), CTL mediatedtarget cell killing, and CTL cytokine release by CTLs.

A CTL-based cytotoxicity assay is used to assess the consequences ofepitope presentation. The assay involves tissue-cultured target cellsand T-cells. Target cells are intoxicated with exemplary cell-targetingmolecules of the invention as described above in Section III. TestingEpitope-Peptide Delivery and Target Cell Surface Presentation etc.Briefly, target positive cells are incubated for twenty hours instandard conditions with different exogenously administered molecules,including a cell-targeting molecule of the invention. Next. CTLs areadded to the treated target cells and incubated to allow for the CTLs torecognize and bind any target cells displaying epitope-peptide/MHC classI complexes (pMHC Is). Then certain functional consequences of pMHC Irecognition are investigated using standard methods known to the skilledworker, including CTL binding to target cells, epitope-presenting targetcell killing by CTL-mediated cytolysis, and the release of cytokines,such as IFN-γ or interleukins by ELISA or ELISPOT.

Assays known to the skilled worker were performed to assess functionalconsequences of intercellular engagement of T-cells in response tocell-surface epitope presentation by targeted cancer cells displayingepitopes delivered by exemplary cell-targeting molecules of the presentinvention. The results from an in vitro intercellular immune cellengagement assay show that Shiga toxin effector polypeptide-mediateddelivery of a fused epitope-peptide to target positive cancer cells andsubsequent cell-surface presentation of the epitope by the targetedcancer cells can result in intercellular engagement of immune cells withfunctional consequences, specifically IFN-γ secretion by PBMCs.

A routine assay known to the skilled worker is performed to assessintercellular T-cell activation after recognition of cell-surfaceepitope presentation by targeted cancer cells displaying an epitopedelivered by a Shiga toxin derived cell-targeting molecule. This invitro T-cell engagement assay shows that Shiga toxin effectorpolypeptide-mediated delivery of a fused epitope-peptide to targetpositive cancer cells and subsequent cell-surface presentation of theepitope by the targeted cancer cells can result in intercellularengagement of T-cells and intracellular cell signaling characteristic ofT-cell activation.

In addition, the activation of CTLs by target cells displayingepitope-peptideiMHC class I complexes (pMHC Is) is quantified usingcommercially available CTL response assays, e.g. CytoTox96®non-radioactive assays (Promega, Madison. Wis., U.S.), Granzyme BELISpot assays (Mabtech, Inc., Cincinnati, Ohio, U.S.), caspase activityassays, and LAMP-I translocation flow cytometric assays. To specificallymonitor CTL-mediated killing of target cells, carboxyfluoresceinsuccinimidyl ester (CFSE) is used to target-cells for in vitro and inviwvo investigation as described in the art (see e.g. Durward M et al.,J Vis Exp 45 pii 2250 (2010)).

In summary, these results show that Shiga toxin effector functions,particularly subcellular routing, can be retained at high levels despitethe presence of a fused epitope-peptide on the carboxy-terminus and thepresence of numerous mutations in the Shiga toxin derived componentproviding de-immunization and protease-cleavage resistance. Furthermore,several cell-targeting molecules exhibit a level of epitope cargodelivery sufficient to produce a level of epitope-MHC class Ipresentation to stimulate intercellular, T-cell engagement withepitope-cargo-presenting cells.

Example 2. Cell-Targeting Molecules Comprising Shiga Toxin A SubunitDerived Polypeptides and Fused, T-Cell Epitope-Peptides CreatingCell-Targeting, Fusion Proteins Comprising Shiga Toxin A SubunitEffector Polypeptide Regions and Fused, T-Cell Epitope-Peptide Regions

Cell-targeting, fusion proteins of this Example comprised acell-targeting binding region polypeptide, a Shiga toxin A Subuniteffector polypeptide, a proteinaceous linker, and a human CD8+ T-cellepitope described in WO 2015/113005, WO 2016/196344, and/orPCT/US2016/043902.

Using techniques known in the art, cell-targeting fusion proteins werecreated by genetically fusing a human CD8+ T-cell epitope-peptide to theamino terminus (N-terminus) or carboxy terminus (C-terminus) of apolypeptide component of a parental, cell-targeting proteincomprising 1) a Shiga toxin A Subunit effector polypeptide and 2) acell-targeting binding region polypeptide separated by a proteinaceouslinker. The fused, CD8+ T-cell epitopes were chosen from among severalT-cell epitope-peptides of proteins originating in viruses that commonlyinfect humans. Certain, cell-targeting, fusion proteins of this Examplewere constructed such that each comprised a single, continuouspolypeptide comprising a cell-targeting, binding region polypeptide, aShiga toxin A Subunit effector polypeptide, and a fused, heterologous,CD8+ T-cell epitope.

The exemplary cell-targeting molecules of the present invention thatwere produced and tested in this Example were SLT-1A-DI-1::scFv8::C2(SEQ ID NO:256), “inactive SLT-1A-DI-4::scFv6::(C2)₃” (SEQ ID NO:253),and “inactive SLT-A-DI-1::scFv8::C2” (SEQ ID NO:254). Othercell-targeting molecules that were produced and tested in this Exampleincluded: C2::SLT-A::scFv2 (SEQ ID NO:267), “inactive C2::SLT-1A::scFv2”(SEQ ID NO:268), SLT-1A::scFv1::C2 (SEQ ID NO:278), SLT-1A::scFv2::C2(SEQ ID NO:269), “inactive SLT-1A::scFv2::C2” (SEQ ID NO:270),F2::SLT-1A::scFv2 (SEQ ID NO:271), scFv3::F2::SLT-1A (SEQ ID NO:272),scFv4::F2::SLT-1A (SEQ ID NO:273), SLT-1A::scFv5::C2 (SEQ ID NO:274),SLT-1A::scFv6::F2 (SEQ ID NO:275). “inactive SLT-1A::scFv6::F2” (SEQ IDNO:276), SLT-1A::scFv7::C2 (SEQ ID NO:277), and C1::SLT-1A::scFv1 (SEQID NO:260), C1-2::SLT-1A::scFv1 (SEQ ID NO:261), C3::SLT-1A::scFv1 (SEQID NO:262), C24::SLT-1A::scFv1 (SEQ ID NO:263), SLT-1A::scFv1::C1 (SEQID NO:268), SLT-1A::scFv1::C24-2 (SEQ ID NO:264), SLT-1A::scFv1::E2 (SEQID NO:265), and SLT-1A::scFv1::F3 (SEQ ID NO:266). These cell-targeting,fusion proteins each comprised a cell-targeting binding regioncomprising a single-chain variable fragment (scFv), a Shiga toxin ASubunit effector polypeptide derived from the A Subunit of Shiga-liketoxin 1 (SLT-1A), and a human CD8+ T-cell epitope-peptide fused toeither the binding region or the Shiga toxin effector polypeptide.

All the Shiga toxin effector polypeptide regions of the cell-targetingmolecules of this Example consisted of or were derived from amino acids1-251 of SLT-1A (SEQ ID NO: 1), and some of them contained two or moreamino acid residue substitutions relative to a wild-type Shiga toxin ASubunit, such as, e.g., the catalytic domain inactivating substitutionEl 67D, C242S, and/or substitutions resulting in furin-cleavageresistance R248A/R251A (see e.g. WO 2015/191764; WO 2016/196344). TheShiga toxin A subunit effector polypeptide component of the exemplarycell-targeting molecules of the invention of this Example includeSLT-1A-DI-1 (SEQ ID NO:30), “inactive SLT-1A-DI-1” (SEQ ID NO:35), and“inactive SLT-1A-DI-4” (SEQ ID NO:38), which may be either catalyticallyactive or modified to have reduced catalytic activity (see e.g. SEQ IDNO: 33). As used in this Example, the cell-targeting moleculenomenclature “inactive” refers to a molecule comprising only those Shigatoxin A Subunit effector polypeptide component(s) that have the E167Dsubstitution. This single amino acid residue substitution can attenuateShiga toxin A Subunit catalytic activity, such as, e.g. by a factor10,000-fold.

The immunoglobulin-type binding regions scFv1, scFv2, scFv3, scFv4,scFv5, scFv6, scFv7, and scFv8 are each single-chain variable fragmentsthat bound with high-affinity to a certain cell-surface, targetbiomolecule physically coupled to the surface of certain human cancercells. Both scFv1 and scFv2 bind with high affinity and specificity tothe same extracellular target biomolecule. Both scFv3 and scFv5 bindwith high affinity and specificity to the same extracellular targetbiomolecule. All three of scFv6, scFv7, and scFv8 bind with highaffinity and specificity to the same extracellular target biomolecule.None of scFv1, scFv3, scFv4, and scFv6 target the same extracellulartarget biomolecule.

All of the cell-targeting molecules tested in the experiments of thisExample, including reference cell-targeting molecules, were produced ina bacterial system and purified by column chromatography usingtechniques known to the skilled worker.

Testing the Shiga Toxin A Subunit Effector Polypeptide Components ofCell-Targeting Molecules for Retention of Shiga Toxin Functions afterthe Fusion of Binding Regions and T-Cell Epitope-Peptides

Cell-targeting proteins were tested for retention of Shiga toxin ASubunit effector functions after the fusion of heterologous, CD8+ T-cellepitope-peptides. The Shiga toxin A Subunit effector functions analyzedwere: catalytic inactivation of eukaryotic ribosomes, cytotoxicity, andby inference self-directing subcellular routing to the cytosol. At leastseven, cell-targeting proteins exhibited catalytic activity comparableto a wild-type, Shiga toxin effector polypeptide not fused to anyheterologous, T-cell epitope-peptide or additional polypeptide moiety.

1. Testing the Ribosome Inhibition Ability of Cell-Targeting Moleculesof the Invention

The catalytic activities of Shiga toxin A Subunit derived Shiga toxineffector polypeptide regions of cell-targeting molecules were testedusing a ribosome inhibition assay.

The ribosome inactivation capabilities of cell-targeting proteins ofthis Example were determined using a cell-free, in vitro proteintranslation assay using the TNT® Quick Coupled Transcription/TranslationKit (L1170 Promega Madison, Wis., U.S.). The kit includes Luciferase T7Control DNA (L4821 Promega Madison, Wis., U.S.) and TNT® Quick MasterMix. The ribosome activity reaction was prepared according tomanufacturer's instructions. A series of 10-fold dilutions of the Shigatoxin derived, cell-targeting protein to be tested was prepared in anappropriate buffer and a series of identical TNT reaction mixturecomponents were created for each dilution. Each sample in the dilutionseries was combined with each of the TNT reaction mixtures along withthe Luciferase T7 Control DNA. The test samples were incubated for 1.5hours at 30 degrees Celsius (° C.). After the incubation. LuciferaseAssay Reagent (E1483 Promega, Madison. W1, U.S.) was added to all testsamples and the amount of luciferase protein translation was measured byluminescence according to manufacturer's instructions.

The level of translational inhibition was determined by non-linearregression analysis of log-transformed concentrations of total proteinversus relative luminescence units. Using statistical software (GraphPadPrism, San Diego, Calif. U.S.), the half maximal inhibitoryconcentration (IC₅₀) value was calculated for each sample using thePrism software function of log(inhibitor) vs, response (threeparameters) [Y=Bottom+((Top−Bottom)/(1+10{circumflex over ( )} (X−LogIC₅₀)))] under the heading dose-response-inhibition. The IC₅₀ values foreach Shiga toxin derived, cell-targeting protein from one or moreexperiments was calculated and is shown in Table 3 in picomolar (pM).Any cell-targeting molecule which exhibited an IC₅₀ within 10-fold of apositive control molecule comprising a wild-type, Shiga toxin effectorpolypeptide (e.g. SLT-1A-WT (SEQ ID NO:279)) is considered herein toexhibit ribosome inhibition activity comparable to wild-type.

TABLE 3 Ribosomal Inhibition by Shiga Toxin Derived, Cell-TargetingProteins Fused to Heterologous Epitope-Peptides ribosomal fusedinhibition Protein epitope fusion location IC₅₀ (pM) Experiment 1C1::SLT-1A::scFv1 C1 N-terminal fusion 3.2 C1-2::SLT-1A::scFv1 C1-2N-terminal fusion 1.2 C3::SLT-1A::scFv1 C3 N-terminal fusion 5.6C24::SLT-1A::scFv1 C24 N-terminal fusion 1.4 SLT-1A::scFv1 none; controlmolecule having no fused epitope 1.2 Experiment 2 C2::SLT-1A::scFv2 C2N-terminal fusion 12.6 SLT-1A::scFv2::C2 C2 C-terminal fusion 13.1SLT-1A::scFv2 none; control molecule having no fused epitope 8.3Experiment 3 F2::SLT-1A::scFv2 F2 N-terminal fusion 2.2 SLT-1A::scFv2none; control molecule having no fused epitope 8.2 Experiment 4scFv3::F2::SLT-1A F2 between binding region and Shiga toxin 6.0 effector(N-terminal of Shiga toxin effector) sc.Fv4::F2::SLT-1A F2 betweenbinding region and Shiga toxin 5.0 effector (N-terminal of Shiga toxineffector) SLT-1A-WT only none; control molecule having no fused epitope9.8 Experiment 5 SLT-1A::scFv5::C2 C2 C-terminal fusion 1.0SLT-1A::scFv5 none; control molecule having no fused epitope 2.1Experiment 6 SLT-1A::scFv6::F2 F2 C-terminal fusion 5.6 SLT-1A::scFv6none; control molecule having no fused epitope 3.2 SLT-1A-WT only none;control molecule having no fused epitope 6.1

As shown in Table 3, cell-targeting proteins exhibited potent ribosomeinhibition comparable to the positive controls: 1) a “SLT-1A-WT only”polypeptide (SEQ ID NO:279) comprising only a wild-type Shiga toxin ASubunit polypeptide sequence and 2) a cell-targeting protein comprisinga SLT-1A derived Shiga toxin effector polypeptide fused to a scFvbinding region but lacking any fused, heterologous, CD8+ T-cellepitope-peptide, e.g., SLT-1A::scFv1 (SEQ ID NO:280), SLT-1A::scFv2 (SEQID NO:281), SLT-1A::scFv5 (SEQ ID NO:283), or SLT-1A::scFv6 (SEQ IDNO:284).

2. Testing the Cytotoxic Activities of Cell-Targeting Molecules of theInvention

The cytotoxic activities of cell-targeting molecules were measured usinga tissue culture cell-based toxicity assay. The concentration ofexogenously administered cell-targeting molecule which kills half thecells in a homogenous cell population (half-maximal cytotoxicconcentration) was determined for certain cell-targeting molecules. Thecytotoxicities of cell-targeting molecules were tested using cell-killassays involving either target biomolecule positive or targetbiomolecule negative cells with respect to the target biomolecule ofeach cell-targeting molecule's binding region.

The cell-kill assays were performed as follows. Human tumor cell linecells were plated (typically at 2×10³ cells per well for adherent cells,plated the day prior to protein addition, or 7.5×10³ cells per well forsuspension cells, plated the same day as protein addition) in 20 μL cellculture medium in 384-well plates. A series of 10-fold dilutions of theproteins to be tested was prepared in an appropriate buffer, and 5 μL ofthe dilutions or only buffer as a negative control were added to thecells. Control wells containing only cell culture medium were used forbaseline correction. The cell samples were incubated with the proteinsor just buffer for 3 or 5 days at 37° C. and in an atmosphere of 5%carbon dioxide (CO₂). The total cell survival or percent viability wasdetermined using a luminescent readout using the CellTiter-Glo®Luminescent Cell Viability Assay (G7573 Promega Madison, W1, U.S.)according to the manufacturer's instructions as measured in relativelight units (RLU).

The Percent Viability of experimental wells was calculated using thefollowing equation: (Test RLU−Average Media RLU)/(Average CellsRLU−Average Media RLU)*100. Log protein concentration versus PercentViability was plotted in Prism (GraphPad Prism, San Diego, Calif., U.S.)and log (inhibitor) versus response (3 parameter) analysis were used todetermine the half-maximal cytotoxic concentration (CD₅₀) value for thetested proteins. The CD₅₀ values for each cell-targeting protein testedwas calculated when possible.

The specificity of the cytotoxic activity of a given cell-targetingmolecule was determined by comparing cell kill activities toward cellsexpressing a significant amount of a target biomolecule of the bindingregion of the cell-targeting molecule (target positive cells) withcell-kill activities toward cells which do not exhibit any significantamount of any target biomolecule of the binding region of thecell-targeting molecule physically coupled to any cellular surface(target negative cells). This was accomplished by determining thehalf-maximal cytotoxic concentrations of a given cell-targeting moleculetoward cell populations which were positive for cell surface expressionof the target biomolecule of the cell-targeting molecule being analyzed,and, then, using the same cell-targeting molecule concentration range toattempt to determine the half-maximal cytotoxic concentrations towardcell populations which were negative for cell surface expression of thetarget biomolecule of the cell-targeting molecule. In some experiments,the target negative cells treated with the maximum amount of theShia-toxin containing molecule did not show any change in viability ascompared to a “buffer only” negative control.

The cytotoxic activity levels of various molecules tested using thecell-kill assay described above are reported in Table 4. As reported inTable 4, cell targeting proteins which were tested in this assayexhibited potent cytotoxicity. While the fusion of a heterologous, CD8+T-cell epitope-peptide to a Shiga toxin derived, cell-targeting proteincan result in no change in cytotoxicity, some cell-targeting proteinsexhibited reduced cytotoxicity as compared to the parental protein fromwhich it was derived, which did not comprise any fused, heterologousepitope-peptide (Table 4). As reported in the Examples, a moleculeexhibiting a CD₅₀ value within 10-fold of a CD₅₀ value measured for areference molecule is considered to exhibit cytotoxic activitycomparable to that reference molecule. In particular, any cell-targetingmolecule that exhibited a CD₅₀ value to a target positive cellpopulation within 10-fold of the CD₅₀ value of a referencecell-targeting molecule comprising the same binding region and awild-type, Shiga toxin effector polypeptide (e.g. SLT-1A-WT (SEQ IDNO:279)) but not comprising any fused, heterologous, T-cellepitope-peptide, toward the same cell-type is referred to herein as“comparable to wild-type.” Cell-targeting molecules that exhibited aCD₅₀ value to a target positive cell population within 100-fold to10-fold of a reference molecule comprising the same binding region andthe same Shiga toxin effector polypeptide but not comprising any fused,heterologous, T-cell epitope-peptide is referred to herein as active but“attenuated.”

TABLE 4 Cytotoxic Activities of Shiga Toxin Derived, Cell-TargetingProteins Comprising Fused, Heterologous Epitope-Peptides fused cell-typein Cytotoxicity Protein epitope fusion location assay CD₅₀ (nM)Experiment 1 C1::SLT-1A::scFv1 C1 N-terminus Cell Line A 0.025 (targetpositive) C1-2::SLT-1A::scFv1 C1-2 N-terminus Cell Line A 0.067 (targetpositive) C3::SLT-1A::scFv1 C3 N-terminus Cell Line A 0.059 (targetpositive) C24::SLT-1A::scFv1 C24 N-terminus Cell Line A 0.240 (targetpositive) SLT-1A::scFv1 none; control molecule having Cell Line A 0.010no fused epitope (target positive) SLT-1A-WT only none; control moleculehaving Cell Line A >100 nM no fused epitope (target positive) Experiment2 SLT-1A::scFv1::C1 C1 C-terminus Cell Line A 0.009 (target positive)SLT-1A::scFv1::C24-2 C24-2 C-terminus Cell Line A 0.263 (targetpositive) SLT-1A::scFv1::F3 F3 C-terminus Cell Line A 0.041 (targetpositive) SLT-1A::scFv1::E2 E2 C-terminus Cell Line A 0.213 (targetpositive) SLT-1A::scFv1 none; control molecule having Cell Line A 0.004no fused epitope (target positive) Experiment 3 SLT-1A::scFv1::C2 C2C-terminus Cell Line B 0.041 (target positive) SLT-1A::scFv1 none;control molecule having Cell Line B 0.097 no fused epitope (targetpositive) SLT-1A-WT only none; control molecule having Cell Line B >100nM no fused epitope (target positive) SLT-1A::scFv1::C2 C2 C-terminusCell Line C >100 nM (target negative) SLT-1A::scFv1 none; controlmolecule having Cell Line C >100 nM no fused epitope (target negative)SLT-1A-WT only none; control molecule having Cell Line C >100 nM nofused epitope (target negative) Experiment 4 F2::SLT-1A::scFv2 F2N-terminus Cell Line A 0.016 (target positive) SLT-1A::scFv2 none;control molecule having Cell Line A 0.016 no fused epitope (targetpositive) SLT-1A-WT only none; control molecule having Cell Line A33.000 no fused epitope (target positive) F2::SLT-1A::scFv2 F2N-terminus Cell Line B 0.0140 (target positive) SLT-1A::scFv2 none;control molecule having Cell Line B 0.0250 no fused epitope (targetpositive) SLT-1A-WT only none; control molecule having Cell Line B310.000 no fused epitope (target positive) Experiment 5C2::SLT-1A::scFv2 C2 N-terminus Cell Line B 0.35 (target positive)SLT-1A::scFv2::C2 C2 C-terminus Cell Line B 0.31 (target positive)inactive C2::SLT-1A::scFv2 C2 N-terminus Cell Line B >100 nM (targetpositive) SLT-1A::scFv2::C2 none; control molecule having Cell Line B0.11 no fused epitope (target positive) SLT-1A-WT only none; controlmolecule having Cell Line B >100 nM no fused epitope (target positive)Experiment 6 scFv3::F2::SLT-1A F2 between binding region Cell Line D1.42 and Shiga toxin effector (target positive) (N-tenninal of Shigatoxin effector) scFv3::SLT-1A none; control molecule having Cell Line D1.35 no fused epitope (target positive) SLT-1A-WT only none; controlmolecule having Cell Line D >100 nM no fused epitope (target positive)Experiment 7 SLT-1A::scFv5::C2 C2 C-terminus Cell Line E 0.33 (targetpositive) SLT-1A::scFv5 none; control molecule having Cell Line E 0.25no fused epitope (target positive) SLT-1A-WT none; control moleculehaving Cell Line E >100 nM no fused epitope (target positive) Experiment8 SLT-1A::scFv6::F2 F2 C-terminus Cell Line F 0.061 (target positive)SLT-1A::scFv6 none; control molecule having Cell Line F 0.142 no fusedepitope (target positive) SLT-1A::scFv7::C2 C2 C-terminus Cell Line F0.011 (target positive) SLT-1A::scFv7 none; control molecule having CellLine F 0.018 no fused epitope (target positive)

All the tested, cell-targeting proteins potently killed target positivecells (Table 4) but did not kill comparable percentages of targetnegative cells at the same dosages (see e.g. FIGS. 6 and 7). FIGS. 6 and7 graphically show the specific cytotoxicity of the cell-targetingprotein SLT-1A::scFv1::C2 (SEQ ID NO:278) was only specific to targetexpressing cells (FIG. 6) but not target negative cells over theconcentration range tested (FIG. 7). The CD₅₀ values of cell-targetingproteins toward target negative cells could not be calculated from theconcentration range of cell-targeting protein tested because an accuratecurve could not be generated when there was not a sizeable decrease incell viability at the highest tested concentrations (see e.g. FIG. 7).

Testing Epitope-Peptide Delivery and Target Cell Surface Presentation ofDelivered Epitope-Peptides

The successful delivery of a T-cell epitope can be determined bydetecting specific cell surface, MHC class I molecule/epitope complexes(pMHC Is). In order to test whether a cell-targeting protein can delivera fused T-cell epitope to the MHC class I presentation pathway of targetcells, an assay was employed which detects human, MHC Class I moleculescomplexed with specific epitopes. A flow cytometry method was used todemonstrate delivery of a T-cell epitope (fused to a Shiga toxin ASubunit derived cell-targeting protein) and extracellular display of thedelivered T-cell epitope-peptide in complex with MHC Class I moleculeson the surfaces of target cells. This flow cytometry method utilizessoluble human T-cell receptor (TCR) multimer reagents (Soluble T-CellAntigen Receptor STAR™ Multimer, Altor Bioscience Corp., Miramar, Fla.,U.S.), each with high-affinity binding to a different epitope-human HLAcomplex.

Each STAR™ TCR multimer reagent is derived from a specific T-cellreceptor and allows detection of a specific peptide-MHC complex based onthe ability of the chosen TCR to recognize a specific peptide presentedin the context of a particular MHC class I molecule. These TCR multimersare composed of recombinant human TCRs which have been biotinylated andmultimerized with streptavidin. The TCR multimers are labeled withphycoerythrin (PE). These TCR multimer reagents allow the detection ofspecific peptide-MHC Class I complexes presented on the surfaces ofhuman cells because each soluble TCR multimer type recognizes and stablybinds to a specific peptide-MHC complex under varied conditions (Zhu Xet al., J Immunol 176: 3223-32 (2006)). These TCR multimer reagentsallow the identification and quantitation by flow cytometry ofpeptide-MHC class I complexes present on the surfaces of cells.

The TCR CMV-pp65-PE STAR™ multimer reagent (Altor Bioscience Corp.,Miramar, Fla., U.S.) was used in this Example. MHC class I pathwaypresentation of the human CMV C2 peptide (NLVPMVATV (SEQ ID NO:21)) byhuman cells expressing the HLA-A2 can be detected with the TCRCMV-pp65-PE STAR™ multimer reagent which exhibits high affinityrecognition of the CMV-pp65 epitope-peptide (residues 495-503,NLVPMVATV) complexed to human HLA-A2 and is labeled with PE.

The target cells used in this Example (target positive cell lines B, D,E, F, G, H, I, J, and K) were immortalized human cancer cells availablefrom the ATCC (Manassas Va., U.S.) or the DSMZ (The Leibniz DeutscheSammlung von Mikroorganismen und Zellkulture) (Braunschweig, Del.)).Using standard flow cytometry methods known in the art, the target cellswere confirmed to express on their cell surfaces both the HLA-A2MHC-Class I molecule and the extracellular target biomolecules of thecell-targeting proteins used in this Example. In some experiments, thehuman cancer cells were pretreated with human interferon gamma (IFN-γ)to enhance expression of human HLA-A2.

Sets of target cells were treated by exogenous administration ofcell-targeting molecules comprising a carboxy-terminal fused, viral,CD8+ T-cell epitope: SLT-IA::scFvI::C2 (SEQ ID NO:278), “inactiveSLT-1A::scFv2::C2” (SEQ ID NO:270), SLT-1A::scFv5::C2 (SEQ ID NO:273),and SLT-1A::scFv7::C2 (SEQ ID NO:277); or were treated by exogenousadministration of a negative-control cell-targeting fusion protein whichdid not comprise any fused, heterologous, viral epitope-peptide(SLT-1A::scFv1 (SEQ ID N0280), SLT-1A::scFv2 (SEQ ID NO:282), “inactiveSLT-1A::scFv2” (SEQ ID NO:281), SLT-1A::scFv5 (SEQ ID NO:283), orSLT-1A::scFv7 (SEQ ID NO:286)). The cell-targeting molecules andreference molecules used in these experiments include both catalyticallyactive, cytotoxic cell-targeting molecules and “inactive” cell-targetingmolecules—meaning all their Shiga toxin effector polypeptide componentscomprised the mutation E167D which severely reduces the catalyticactivity of Shiga toxin A Subunits and Shiga toxins. These treatmentswere at cell-targeting molecule concentrations similar to those used byothers taking into account cell-type specific sensitivities to Shigatoxins (see e.g. WO 2015/113005). The treated cells were then incubatedfor 4-16 hours in standard conditions, including at 37° C., and anatmosphere with 5% carbon dioxide, to allow for intoxication mediated bya Shiga toxin effector polypeptide. Then the cells were washed andincubated with the TCR CMV-pp65-PE STAR™ multimer reagent to “stain” C2peptide-HLA-A2 complex-presenting cells.

As controls, sets of target cells were treated in three conditions: 1)without any treatment (“untreated”) meaning there was addition of onlybuffer to the cells and no addition of any exogenous molecules, 2) withexogenously administered CMV C2 peptide (CMV-pp65, aa495-503: sequenceNLVPMVATV (SEQ ID NO:21), synthesized by BioSynthesis, Lewisville, Tex.,U.S.), and/or 3) with exogenously administered CMV C2 peptide ((SEQ IDNO:21), as above) combined with a Peptide Loading Enhancer (“PLE,” AltorBiosicence Corp., Miramar, Fla., U.S.). The C2 peptide (SEQ ID NO:21)combined with PLE treatment allowed for exogenous peptide loading andserved as a positive control. Cells displaying the appropriate MHC classI haplotype can be forced to load the appropriate exogenously appliedpeptide from an extracellular space (i.e. in the absence of cellularinternalization of the applied peptide) or in the presence of PLE, whichis a mixture of B2-microglobulin and other components.

After the treatments, all the sets of cells were washed and incubatedwith the TCR CMV-pp65-PE STAR™ multimer reagent for one hour on ice. Thecells were washed and the fluorescence of the samples was measured byflow cytometry using an Accuri™ C6 flow cytometer (BD Biosciences, SanJose, Calif., U.S.) to detect the presence of and quantify any TCRCMV-pp65-PE STAR™ multimer bound to cells in the population (sometimesreferred to herein as “staining”) in relative light units (RLU).

Table 5 and FIGS. 8-12 show results from experiments using the TCR STAR™assay detecting cell-surface complexes of C2 epitope/HLA-A2 MHC class Imolecule. For each experiment, the untreated control sample was used toidentify the positive and negative cell populations by employing a gatewhich results in less than 1% of cells from the untreated control in the“positive” gate (representing background signal). The same gate was thenapplied to the other samples to characterize the positive population foreach sample. Positive cells in this assay were cells which were bound bythe TCR-CMV-pp65-PE STAR™ reagent and counted in the positive gatedescribed above. In FIG. 8 and FIGS. 10-12, the flow cytometryhistograms are given with the counts (number of cells) on the Y-axis andthe relative fluorescent units (RFU) representing TCR CMV-pp65 STAR™multimer, PE staining signal on the X-axis (log scale). The black lineshows the results for the untreated-cells-only sample, and the gray lineshows the results for the negative controls (treatment with only aparental, cell-targeting protein lacking any viral epitope-peptide), orthe treatment with a specific, cell-targeting protein. In FIGS. 8, 11,and 12, the top panels show the results for the untreated cell samplesusing black lines and the results for the cell-targeting moleculetreated samples using gray lines. In FIGS. 8, 11, and 12, the bottompanels show the results for untreated cell samples using black lines andthe results for the control proteins, which did not comprise any fusedepitope-peptide, using gray lines. In FIG. 10, the top panel shows theresults from a 4-hour incubation and the bottom panel shows the resultsfor a 16-hour incubation. In Table 5, the percentage of cells in atreatment set which stained positive for the C2-epitope-peptide-HLA-A2MHC class I molecule complex is given. Table 5 also shows thecorresponding indexed, mean, fluorescent intensity (“iMFI,” thefluorescence of the positive population multiplied by the percentpositive) in RFU for each treatment set.

TABLE 5 Detection of Cell Surface, MHC Class I/C2 Epitope Complexesafter Delivery of C2 Epitope-Peptides by Cell-Targeting Proteins:Peptide-epitope C2/MHC class I complexes detected on the surfaces ofintoxicated, target cells target incubation percentage of positiveduration pMHC I complex iMFI Protein cell-type (hours) presenting cells(RFU) Experiment 1 SLT-1A::scFv1::C2 Cell Line B  4 hours 33.0% 440SLT-1A::scFv2 Cell Line B  4 hours 5.0% 80 Experiment 2SLT-1A::scFv1::C2 Cell Line B 16 hours 95.4% 28,800 SLT-1A::scFv1 CellLine B 16 hours 5.0% 154 Experiment 3 inactive SLT-1A::scFv2::C2 CellLine G 24 hours 43.3% 4,034 inactive SLT-1A::scFv2 Cell Line G 24 hours0.2% 17 C2 peptide Cell Line G 24 hours 57.8% 5,114 C2 peptide + PLECell Line G 24 hours 0.5% 79 Experiment 4 inactive SLT-1A::scFv2::C2Cell Line B 16 hours 80.5% 3,170 inactive SLT-1A::scFv2 Cell Line B 16hours 4.1% 63 inactive SLT-1A::scFv2::C2 Cell Line H 16 hours 67.9%2,550 inactive SLT-1A::scFv2 Cell Line H 16 hours 3.5% 47 Experiment 5SLT-1A::scFv5::C2 Cell Line E 24 hours 41.9% 17,846 SLT-1A::scFv5 CellLine E 24 hours 0.5% 64 C2 peptide Cell Line E 24 hours 2.4% 357 C2peptide + PLE Cell Line E 24 hours 93.2% 42,429 Experiment 6SLT-1A::scFv7::C2 Cell Line F 16 hours 27.6% 6,132 SLT-1A::scFv7 CellLine F 16 hours 1.7% 365

As seen in Table 5 and FIGS. 8-12, cell samples treated with exemplarycell-targeting proteins displayed expression of the C2-epitope/HLA-A2MHC class I molecule complex on the surfaces of a majority of thetreated cells depending on the incubation duration. Cells treated withthe exogenous cell-targeting proteins SLT-1A::scFv1::C2 (SEQ ID NO:278)or “inactive SLT-1A::scFv2::C2” (SEQ ID NO:270), SLT-1A::scFv5::C2 (SEQID NO:274), and SLT-1A::scFv7::C2 (SEQ ID NO:277) showed a positivesignal for cell-surface, C2-epitope/HLA-A2 complexes on 33-95% of thecells in the samples analyzed (Table 5). In contrast, cells that weretreated with parental cell-targeting proteins, which did not contain anyfused T-cell epitope-peptide as a negative control, exhibited positivecell staining of five percent or less of the cells in the treated cellpopulation (Table 5; FIGS. 8-12).

While the majority of cells treated with cell-targeting proteins of thisExample displayed on a cell surface the C2-epitope/HLA-A2 complex, fivepercent or less of the cells in “untreated” cell populations displayedTCR STAR™ staining for C2-epitope/HLA-A2 complexes (Table 5: FIG. 8:FIG. 10). The positive control treatment showed robust staining of 99%of the cells in the population due exclusively to loading of onlyexogenous C2 epitope-peptide (SEQ ID NO:21) in the presence of thepeptide loading enhancer (FIG. 9). Due to processing efficiency andkinetics, which were not measured, it is possible that the percentage ofpresented C2-epitope/HLA-A2 complexes detected at a single time-point ina “cell-targeting protein” treatment sample may not accurately reflectthe maximum quantity of C2-epitope/HLA-A2 presentation possible afterdelivery by a given cell-targeting protein.

The detection of the T-cell epitope C2 (SEQ ID NO:21) complexed withhuman MHC Class I molecules (C2 epitope-peptide/HLA-A2) on the cellsurface of cell-targeting molecule treated target cells demonstratedthat cell-targeting proteins (SLT-1A::scFv1::C2 (SEQ ID NO:278),“inactive SLT-1A::scFv2::C2” (SEQ ID NO:270), SLT-1A::scFv5::C2 (SEQ IDNO:274), and SLT-1A::scFv7::C2 (SEQ ID NO:277)) comprising this fusedepitope-peptide (C2 (SEQ ID NO:21)) were capable of entering targetcells, performing sufficient sub-cellular routing, and deliveringsufficient C2 (SEQ ID NO:21) epitope to the MHC class I pathway forsurface presentation by target cell surface.

Testing Cytotoxic T-Cell Mediated Cytolysis of Intoxicated Target Cellsand Other Immune Responses Triggered by MHC Class I Presentation ofT-Cell Epitopes Delivered by Cell-Targeting Molecules of the PresentInvention

In this Example, standard assays known in the art are used toinvestigate the functional consequences of target cells' MHC class Ipresentation of T-cell epitopes delivered by exemplary cell-targetingmolecules. The functional consequences to investigate include CTLactivation (e.g. signal cascade induction). CTL mediated target cellkilling, and CTL cytokine release by CTLs.

A CTL-based cytotoxicity assay is used to assess the consequences ofepitope presentation. The assay involves tissue-cultured target cellsand T-cells. Target cells are intoxicated with exemplary cell-targetingmolecules as described above in this Example in the sub-section “TestingEpitope-Peptide Delivery and Target Cell Surface Presentation” etc.Briefly, target positive cells are incubated for twenty hours instandard conditions with different exogenously administered molecules,including a cell-targeting molecule. Next, CTLs are added to the treatedtarget cells and incubated to allow for the CTLs to recognize and bindany target cells displaying epitope-peptide/MHC class I complexes (pMHCIs). Then certain functional consequences of pMHC I recognition areinvestigated using standard methods known to the skilled worker,including CTL binding to target cells, epitope-presenting target cellkilling by CTL-mediated cytolysis, and the release of cytokines, such asIFN-γ or interleukins by ELISA or ELISPOT.

Assays were performed to assess functional consequences of intercellularengagement of T-cells in response to cell-surface epitope presentationby targeted cancer cells displaying epitopes delivered by cell-targetingmolecules.

FIG. 13 and Table 6 show the results of an Interferon Gamma ELIspotassay (Mabtech, Inc., Cincinnati, Ohio, U.S.) used according tomanufacturer's instructions. This ELISPOT assay can be used to quantifyIFN-γ secretion as each spot indicates a IFN-γ secreting cell. Briefly,samples of cells of target positive cell line G were incubated for 20hours with either just phosphate buffered saline (PBS) buffer (“bufferonly”), “inactive SLT-1A::scFv2::C2” (SEQ ID NO:270), or the referencemolecule “inactive SLT-1A::scFv2” (SEQ ID NO:282). The samples werewashed with PBS and added to ELISPOT plates already loaded with humanPBMCs (HLA-A2 serotype) from Cellular Technology Limited (ShakerHeights, Ohio, U.S.). The plates were incubated for an additional 24hours, and then spots were detected according to the Interferon GammaELIspot assay Mabtech kit instructions and quantified using an ELISPOTplate reader (Zellnet Consulting, Inc., Fort Lee, N.J., U.S.).

TABLE 6 Interferon Gamma Secretion by PBMCs after Recognizing EpitopePresentation by Target Cells Incubated with “inactiveSLTA-1A::scFv2::C2” target Average positive number of Average AreaProtein cell-type spots per spot inactive SLT-1A::scFv2::C2 Cell Line G490 2,636,291 inactive SLT-1A::scFv2 Cell Line G 280 1,511,726 bufferonly Cell Line G 334 2,144,217

The results in Table 6 and FIG. 13 show that the incubation of cell lineG cells with the cell-targeting molecule “inactive SLT-1A::scFv2::C2”(SEQ ID NO:270) resulted in a PBMC luciferase activity signal greaterthan the background signal determined using the buffer only treated cellsample or the luciferase signal from the sample cells treated with thereference molecule “inactive SLT-1A::scFv2” (SEQ ID NO:282). The resultsfrom this in vitro intercellular immune cell engagement assay showedthat Shiga toxin effector polypeptide-mediated delivery of a fusedepitope-peptide to target positive cancer cells and subsequentcell-surface presentation of the epitope by the targeted cancer cellscan result in intercellular engagement of immune cells with functionalconsequences, specifically IFN-γ secretion by PBMCs.

When an effector T-cell recognizes a specific epitope-MHC-I complex, theT-cell may initiate an intracellular signaling cascade that drives thetranslocation of nuclear factor of activated T-cells (NFAT)transcription factors from the cytosol into the nucleus and can resultin the stimulation of the expression of genes that contain a NFATresponse element(s) (RE) (see e.g. Macian F, Nat Rev Immunol 5: 472-84(2005)). A J76 T-cell line engineered to express a human T-cell receptorthat specifically recognizes the F2 peptidehuman HLA A2 MHC class Imolecule complex (Berdien B et al., Hum Vaccin Immunother 9: 1205-16(2013)) was transfected with a luciferase expression vector(pGL4.30[Iuc2P/NFAT-RE/Hygro], CAT # E8481, Promega Corp., Madison,Wis., U.S.) that is regulated by an NFAT-RE. When theluciferase-reporter-transfected J76 TCR specific cell recognizes a celldisplaying the HLA-A2/F2 epitope-peptide (SEQ ID NO:25) complex, thenexpression of luciferase can be stimulated by NFAT transcription factorsbinding to the NFAT-RE of the expression vector. Luciferase activitylevels in the transfected J76 cells can be quantified by the addition ofa standard luciferase substrate and then reading luminescence levelsusing a photodetector.

An assay was performed to assess intercellular T-cell activation afterrecognition of cell-surface epitope presentation by targeted cancercells displaying an epitope delivered by a cell-targeting molecule.Briefly, cells samples of cell line F were incubated with “inactiveSLT-1A::scFv6::F2” (SEQ ID NO:276), the reference molecule “inactiveSLT-1A::scFv6” (SEQ ID NO:285), or just buffer alone for 6 hours, andthen washed. Then, luciferase-reporter-transfected J76 T-cells weremixed with each sample, and the mixtures of cells were incubated for 18hours. Next, luciferase activity was measured using the One-Glo™Luciferase Assay System reagent (Promega Corp., Madison, Wis., U.S.).FIG. 14 and Table 7 shows the results from this intercellular T-cellengagement assay.

TABLE 7 Luciferase Signal Driven by the NFAT Response Element inReporter Cells after Recognition of Epitope Presentation by Target CellsIncubated with “inactive SLTA-1A::scFv6::F2” target positive AverageLuciferase Signal Protein cell-type (RLU) inactive SLT-1A::scFv6::F2Cell Line F 565 inactive SLT-1A::scFv6 Cell Line F 259 buffer only CellLine F 242

The results in Table 7 and FIG. 14 show that incubation with thecell-targeting molecule “inactive SLT-1A::scFv6::F2” (SEQ ID NO:276)resulted in luciferase activity levels greater than the backgroundluciferase activity signal determined using “buffer only” treated cellsor the luciferase activity from cell samples treated with the negativecontrol molecule “inactive SLT-1A::scFv6” (SEQ ID NO:285). This in vitroT-cell engagement assay showed that Shiga toxin effectorpolypeptide-mediated delivery of a fused epitope-peptide to targetpositive cancer cells and subsequent cell-surface presentation of theepitope by the targeted cancer cells can result in intercellularengagement of T-cells and an intracellular cell signaling readoutcharacteristic of T-cell activation.

When an effector T-cell recognizes a specific epitope-MHC-I complex, theT-cell may initiate an intracellular signaling cascade that promoteseffector cytokine (e.g. IFN-γ) secretion and/or results in inintercellular, immune cell-mediated killing of the cell presenting thatspecific epitope MHC-I complex. An assay was performed to assess T-cellsecretion of IFN-γ and CD8+ T-cell-mediated cytotoxicity afterrecognition of cell-surface epitope presentation by targeted cancercells displaying an epitope delivered by a cell-targeting molecule. Thisassay involves the coincubation of tumor cells, wvhich were pretreatedwith a cell-targeting molecule of the present invention, with peripheralblood mononuclear cells (PBMCs), including T-cells capable ofrecognizing a specific pMHC I, i.e. T-cells expressing a T-cell receptorthat specifically recognizes the peptide-MHC class I molecule complex onthe surface of another presenting cell.

The coincubation intercellular T-cell assay was performed as follows tomeasure IFN-γ secretion and target cell killing. Cells of cell line I,which is target positive for the extracellular molecule bound by scFv6,were incubated for four hours with either 500 nM of “inactiveSLT-1A-DI-4::scFv6::(C2);” (SEQ ID NO:253) or PBS alone (“buffer only”)at 37° C., and in an atmosphere of five percent CO₂. Then the cells werewashed and combined with media containing PBMCs which are HLA-A02/C2seropositive. Prior to coculture, the PBMCs were enriched forC2-restricted T-cells by one to two weeks of culture-expansion in thepresence of the C2 peptide (SEQ ID NO:21) to obtain a frequency of aboutone to five percent C2-reactive T-cells (these PBMCs are herein referredto as “C2-restricted PBMCs”). The targeted tumor cells and C2-restrictedPBMCs were co-incubated for 24 hours at a ratio of five PBMCs to onetarget positive tumor cell (5:1) at 37° C., and in an atmosphere of fivepercent CO₂. Supernatants were harvested and IFN-γ concentrations weremeasured using a cytokine-specific IFN-γ ELISA Kit (Biolegend, Inc., SanDiego, Calif., U.S.), according to manufacturer's instructions. Inaddition, after harvesting ofsupernatants, adherent target positivetumor cells were washed to remove PBMCs, and cell viability of theremaining adherent cells was assessed by CellTiter-Glo, Luminescent CellViability Assay (G7573 Promega Madison, Wis., U.S.), according to themanufacturer's instructions. FIGS. 15 and 16 show the results of thesecoincubation assays of cell-targeting-molecule-treated cell line I andHLA-A02/C2-peptide complex-detecting PBMCs (C2-restricted PBMCs) toassay intercellular T-cell recognition and engagement by measuring IFN-γsecretion and cell viability of tumor cells.

FIG. 15 shows results from the IFN-γ ELISA assay. The results show thatcoculture of tumor target cells (cell line I) with C2-restricted PBMCsafter incubation with the cell targeting molecule “inactiveSLT-1A-DI-4::scFv6::(C2)₃” (SEQ ID NO:253) but not with “buffer only”results in the activation of T-cells among the PBMCs as demonstrated bythe detection of IFN-γ secretion.

FIG. 16 shows results from the CellTiter-Glo, Luminescent Cell ViabilityAssay as measured in RLU. The results show that coculture of tumortarget cells (cell line 1) with C2-restricted PBMCs after incubationwith the cell targeting molecule “inactive SLT-1A-DI-4::scFv6::(C2)₃”(SEQ ID NO:253) but not with “buffer only” results in the activation ofcytotoxic T lymphocytes among the PBMCs and target cell-killing asdemonstrated by the reductions in the viability percentages for theadherent tumor target cells.

The data from the IFN-γ ELISA and CellTiter-Glo® Luminescent CellViability assays demonstrate that Shiga toxin effectorpolypeptide-mediated delivery of a fused epitope-peptide totarget-positive tumor cells and the subsequent cell-surface presentationof the epitope by the tumor cells can result in the activation ofspecific T-cells to release effector cytokines and cause death of targettumor cells (see FIGS. 15-16).

Another coculture tumor cell viability assay was performed where the E:Tratio is kept constant at 5:1 and the concentration of thecell-targeting molecule was varied or the PBMCs were enriched forC2-restricted PBMCs. The cells used were as above with the target cellsbeing tumor cells of cell line I and the PBMCs being C2-restricted. Theresults were compared to the buffer-only control. The targeted tumorcells and C2-restricted PBMCs were co-incubated for 24 hours at 37° C.,and in an atmosphere of five percent CO2. Adherent target positive tumorcells were washed to remove PBMCs and the CellTiter-Glo® LuminescentCell Viability Assay was performed as described above. Table 8 shows theresults of this coculture tumor cell viability assay where theadministered “inactive SLT-1A-DI-4::scFv6::(C2)₃” (SEQ ID NO:253)concentration tested was varied in a step-down fashion among 2 gM, 0.5μM, and 0.125 μM. The data is presented as reduction in the percent ofviability of adherent cells as measured by the IncuCyte® S3 Live-CellAnalysis System (EssenBioscience, Ann Arbor, Mich., U.S.) normalized totime-point zero (baseline viability). For IncuCyte® S3 Live-Cell imagingstudies, cells were plated in standard 96-well tissue culture plates andcultured under standard conditions. Data was obtained from up to fourimages per well as readout by phase, red and green fluorescence viastandard protocols provided by the manufacturer. Comparisons in thissample include the inclusion of PBMCs at an E:T of 5:1 that were eitherculture-expanded for one week prior to coculture with relevant C2peptide (SEQ ID NO:253) or were culture-expanded for one week prior tococulture with relevant C2 peptide (SEQ ID NO:21).

TABLE 8 Intercellular Engagement of Immune Cells Recognizing C2 EpitopePresentation by Target Cells Contacted with “InactiveSLT-1A-DI-4::scFv6::(C2)3” Results in Target Cell Killing in aDose-Dependent Manner Reduction in Viability of Inactive SLTA-1A-DI-Cells in Presence of 4::scFv6::(C2)3 C2-Restricted PBMCs concentration(μM) (% of T = 0) 0.000 0 0.125 37 0.500 50 2.000 68

The data in Table 8 demonstrates that the quantity of target tumor celldeath induced by a cell-targeting molecule of the present invention isdependent on the concentration of cell-targeting molecule administeredto the target tumor cells and the enrichment of PBMCs. The coculture oftarget positive tumor cells pretreated with “inactiveSLT-1A-DI-4::scFv6::(C2)3” (SEQ ID NO:253) and C2-restricted PBMCsresulted in reductions in adherent, target tumor cell viabilitypercentages from about 35 to 65 percent. At cell-targeting moleculeconcentrations as low as 0.125 μM, over 35 percent of the adherenttarget cells were killed in the C2-restricted PBMC-sample. This effectwas dose-dependent as cell-targeting molecule concentrations of 0.5 pMand 2 pM induced a reduction in cell viability of about 50 and 65percent, respectively. The results in Table 8 demonstrate that theconcentration of cell-targeting molecule administered to the targetcells affects the overall T-cell activation and subsequent killing oftargeted tumor cells.

In the above experiments, the ratio of PBMCs to tumor cells was 5 to 1.Additional experiments were performed where this PBMC effector cell totarget cell (“E:T”) ratio was varied. Target tumor cells (cell line I)were incubated with “inactive SLT-1A-DI-4::scFv6::(C2)₃” (SEQ ID NO:253)at a concentration of 500 nM for four hours at 37° C., and in anatmosphere of five percent CO₂ followed by coculture with PBMCs atvarious E:T ratios) for over three days at 37° C., and in an atmosphereof five percent CO₂. Table 9 shows the results for cell viability ofthis assay at 80 hours post coculture as measured by confluence in theIncuCyte® S3 Live-Cell Analysis System. Data is presented as percentviability in relation to the buffer-only control at the assay endpoint.Comparisons are shown for conditions in the context of treatment with afixed dose of cell-targeting molecule or “buffer only” in the presenceof variable E:T ratios of PBMCs to target cells (E:T=5:1, 1:1, 05:1, and0.1:1).

TABLE 9 PBMCs Recognizing C2 Epitope Presentation by Target CellsContacted with “Inactive SLT-1A-DI-4::scFv6::(C2)3” Results in TargetCell Killing in an E:T-Dependent Manner Treatment Inactive SLTA-1A-DI-Effector (E) to 4::scFv6::(C2)3 Buffer (negative control) Target (T)Ratio Reduction in Viability of Cells Reduction in Viability of Cells(E:T) (% of “buffer only”) (% of “buffer only”) 5.0:1.0 64 18 1.0:1.0 3513 0.5:1.0 38 0 0.1:1.0 0 0

The data in Table 9 demonstrates that the quantity of target tumor celldeath induced by a cell-targeting molecule of the present invention isdependent on the E:T ratio. Table 9 reports an observed reduction inviability of adherent cells of at least 60 percent at an E:T ratio of5:1. Thus, more than 60 percent of the adherent target cells were killedafter 80 hours. At more reduced ratios of PBMCs, the less adherent cellswere killed after 80 hours. At lower E:T ratios tested (1:1 and 0.5:1),less than 40 percent of the adherent target cells were killed. For thebuffer-only control, less than 20 percent of the adherent target cellswere killed at the highest E:T ratio of 5:1. The results in Tables 8 and9 demonstrate that both the ratio of PBMCs to target tumor cells and theconcentration of cell-targeting molecule administered to the targettumor cells contribute to the mechanism of tumor cell cytotoxicityinduced by incubation with a cell-targeting molecule of the presentinvention, with high amounts of both cell-targeting molecule and/orPBMCs correlating with increased target cell death.

Antigen specific T-cell activation is associated with increasedinteractions with neighboring cells and immune cell clusteringphenotypes (see e.g. Butz E, Bevan M, Immunity 8: 167-75 (1998)). Toinvestigate this, another coincubation intercellular T-cell assay wasperformed as follows to measure lymphocyte clustering in response totarget cells presenting the delivered C2 epitope complexed with MHCclass I molecules. Target cells (cell line I) were incubated with 500 nMof “inactive SLT-1A-DI-4::scFv6::(C2)3” (SEQ ID NO:253) for 24 hours andthen co-incubated with C2-restricted PBMCs as described above. For theseexperiments, PBMC effectors from among the PBMCs were pre-labeled withcarboxyfluorescein succinimidyl ester (CFSE), a membrane permeable dyeused to track cell numbers and positions within the culture wells. Aftercoincubation, individual PBMCs in the culture wells were imaged everyfour to six hours using the IncuCyte® S3 Live-Cell Analysis System(EssenBioscience, Ann Arbor, Mich., U.S.). The total number of CFSEpositive cells and mean-intensity of CFSE signal were calculated via theincuCyte, S3 software suite using the manufacturer's protocols. Immunecell clusters (CFSE-positive cell clusters of greater than 100 micron)were plotted in Prism (GraphPad Prism. San Diego, Calif., U.S.) andresults of these experiments are shown in FIG. 17.

FIG. 17 shows the results of coincubation intercellular engagementassays with target cell line I cells and C2-restricted PBMCs as measuredby lymphocyte clustering. The results in FIG. 17 show that incubation oftumor cells with the cell targeting molecule “inactiveSLT-1A-DI-4::scFv6::(C2)3” (SEQ ID NO:253) but not “buffer only” resultsin the activation of cell clustering among PBMCs as measured by theaggregation of cells in clusters of greater than 100 microns in size. Inaddition, these PBMC cell clusters show prolonged persistence,demonstrating that treatment with a cell-targeting molecule of thepresent invention can promote long-term T-cell engagement upon coculture(see FIG. 17 showing persistence durations of at least 140 hours).

Much of the previous experimental data is based on the exemplarycell-targeting molecule “inactive SLT-1A-DI-4::scFv6::(C2)₃” (SEQ IDNO:253). Here, are reported experimental results from testing anotherexemplary cell-targeting molecule: “inactive SLT-1A-DI-::scFv8::C2” (SEQID NO:254).

A coincubation intercellular T-cell assay was performed as follows toquantitate the induction of intercellular T-cell engagement (e.g. targettumor cell killing) induced by treating tumor cells with “inactiveSLT-1A-DI-1::scFv8::C2” (SEQ ID NO:254). Cells of cell line I, J, or K,each of which is target positive for the extracellular molecule bound byscFv8, were incubated for four hours with either 100 nM or 500 nM of“inactive SLT-1A-DI-1::scFv8::C2” (SEQ ID NO:254) or with “buffer only”at 37° C., and in an atmosphere of five percent CO₂. Then the cells werewashed and combined with media containing “C2-restricted PBMCs”. Thetargeted tumor cells and C2-restricted PBMCs were co-incubated for 24hours at a ratio of five C2-restricted PBMCs to one target positivetumor cell (5:1,) at 37° C., and in an atmosphere of five percent COz.Supematants were harvested and IFN-γ concentrations were measured usinga cytokine-specific IFN-γ ELISA Kit (Biolegend. Inc., San Diego, Calif.,U.S.), according to manufacturer's instructions. After harvestingofsupernatants, adherent target positive tumor cells were washed toremove PBMCs, and cell viability of the remaining adherent cells wasassessed by CellTiter-Glo® Luminescent Cell Viability Assay (G7573Promega Madison, Wis., U.S.) in RLU, according to the manufacturer'sinstructions. As a control, parallel experiments were performed with 100nM and 500 nM the cell-targeting molecule “inactive SLT-A-DI-::scFv8(SEQ ID NO:256), which lacks any C2 epitope (SEQ ID NO:21) component butotherwise shares 100% identity with “inactive SLT-1A-DI-1::scFv8::C2”(SEQ ID NO:254).

FIGS. 18 and 19 show the results of these coincubation assays usingtarget positive tumor cell lines I, J, and K and using cell-targetingmolecules tested at different concentrations. The results show thatincubating target positive tumor cells with “inactiveSLT-1A-DI-1::scFv8::C2” (SEQ ID NO:254) and the coincubation withC2-restricted PBMCs results in T-cell activation, as measured by IFN-γsecretion (FIG. 18), and induces tumor cell killing via PBMCs (FIG. 19),with both effects occurring in a cell-targeting moleculeconcentration-dependent manner. The results show that neither “bufferonly” or the negative control cell-targeting molecule lacking the of C2epitope-peptide “inactive SLT-1A-DI-1::scFv8” (SEQ ID NO:256) wascapable of inducing IFN-γ secretion after 48 hours post-coculture (FIG.18) or target cell death during 96 hours of coculture (FIG. 19)regardless of the dose tested. Further, these data demonstrate theability of one exemplary cell-targeting molecule of the presentinvention to successfully deliver a CD8+ T-cell epitope for MHC Ipresentation to a range of different tumor cell-types such thatpresentation results in intercellular T-cell engagement and targetcell-killing via the action of targeted T-cells.

Much of the previous experimental data is based on “inactive” forms ofexemplary cell-targeting molecules of the present invention. Here, arereported experimental results from testing an “active form” of anexemplary cell-targeting molecule of the present invention. An activecell-targeting molecule is used herein in the Examples to refer to acell-targeting molecule which comprises a Shiga toxin effectorpolypeptide that is catalytically active. The exemplary cell-targetingmolecule SLT-1A-DI-1::scFv8::C2 (SEQ ID NO:255) is very closely relatedto “inactive SLT-1A-DI-1::scFv8::C2” (SEQ ID NO:254) in that only oneamino acid is different from the amino acid substitution E168D (themolecules share 99.8% identity overall and 99.6% identity between theirShiga toxin effector polypeptide components). SLT-1A-DI-1::scFv8::C2(SEQ ID NO:255) is considered active herein with regard to its Shigatoxin A Subunit effector polypeptide which has catalytically activitiescomparable to a wild-type Shiga toxin A1 fragment and can kill a cell inwhich it is present.

A coincubation intercellular T-cell assay was performed as describedabove using active SLT-1A-DI-::scFv8::C2 (SEQ ID NO:255) to quantitateintercellular T-cell engagement (e.g. target tumor cell killing andT-cell activation) induced by treating tumor cells with an activecell-targeting molecule of the present invention. Briefly, cells of cellline I were pre-incubated for four hours with “buffer alone” or with 30or 100 nM of cell-targeting molecule SLT-1A-DI-1::scFv8::C2 (SEQ IDNO:255) and then cocultured with C2-restricted PBMCs for 48-96 hours at37° C. and in an atmosphere of five percent CO₂. As a negative control,parallel experiments were performed with 30 or 100 nM of thecell-targeting molecule SLT-1A-DI-1::scFv8 (SEQ ID NO:257), which lacksany C2 epitope (SEQ ID NO:21) component but otherwise shares 100%identity with SLT-1A-DI-1::scFv8::C2 (SEQ ID NO:255). FIG. 20 shows theresults for the coincubation assays.

Supernatants were harvested after 48 hours of coculture and IFN-γconcentrations were measured using the cytokine-specific IFN-γ ELISAassay as described above. FIG. 20 shows the results from the IFN-γ ELISAassay. After 96 hours of coculture, adherent target positive tumor cellswere washed to remove PBMCs, and cell viability of the remainingadherent cells was assessed using the CellTiter-Glo@ Luminescent CellViability assay as described above. FIG. 20 shows the results from theadherent cell viability assay as measured by confluence as in RLU usingthe IncuCyte % S3 Live-Cell Analysis System as described above.

The results shown in FIG. 20 show that a catalytically active form of acell-targeting molecule of the present invention can function similar toan inactive cell-targeting molecule in promoting T-cell activation asmeasured by inducing IFN-γ secretion and immune-cell-dependent targetcell killing beyond its more direct mechanism of action: cell-kill viaribosome inhibition and induction of programmed cell death. Theseresults demonstrate the ability of exemplary cell-targeting molecules ofthe present invention to promote immune activation and to induce targettumor cell-killing via at least two distinct mechanisms of action, whichmay function cooperatively to induce more target cell death in thepresence of MHC class I epitope-specific restricted PBMCs.

In addition, the activation of CTLs by target cells displayingepitope-peptide/MHC class I complexes (pMHC Is) is quantified usingcommercially available CTL response assays, e.g. CytoTox96non-radioactive assays (Promega, Madison, Wis., U.S.), Granzyme BELISpot assays (Mabtech. Inc., Cincinnati, Ohio, U.S.), caspase activityassays, and LAMP-1 translocation flow cytometric assays. To specificallymonitor CTL-mediated killing of target cells, carboxyfluoresceinsuccinimidyl ester (CFSE) is used to target-cells for in vitro and invivo investigation as described in the art (see e.g. Durward M et al., JVis Exp 45 pii 2250 (2010)).

Example 3. Melanoma Cell-Targeting Molecules Comprising Shiga Toxin ASubunit Effector Polypeptides and CD8+ T-Cell Epitope-Peptides

In this Example, the Shiga toxin effector polypeptide is derived fromthe A subunit of Shiga-like Toxin 1 (SLT-1A) as described above withamino acid residue substitutions conferring de-immunization (see e.g. WO2015/113005; WO 2015/113007; WO 2016/196344), CD8+ T-cellhyperimmunization (see e.g. WO 2015/113005; WO 2016/196344), andfurin-cleavage resistance, such as, e.g., R248A/R251A (WO 2015/191764:WO 2016/196344). A human, CD8+ T-cell epitope-peptide is selected basedon MHC I molecule binding predictions, HLA types, already characterizedimmunogenicities, and/or readily available reagents as described above,such as the C1-2 epitope-peptide GLDRNSGNY (SEQ ID NO:20). Aproteinaceous binding region is inserted into the SLT-1A effectorpolypeptide which binds melanoma cells (see e.g. Cheung M et al., MolCancer 9: 28 (2010)).

Construction and Production of Melanoma Cell-Targeting Fusion Proteins

The Shiga toxin effector polypeptide and the CD8+ T-cell epitope arefused together to form a single, continuous polypeptide, such that theCD8+ T-cell epitope is not embedded or inserted in the Shiga toxineffector polypeptide.

Determining the In Vitro Characteristics of Melanoma Cell-TargetingMolecules

The binding characteristics of cell-targeting molecules of this Examplefor melanoma cells is determined by fluorescence-based, flow-cytometry.The Bmax for certain melanoma cell-targeting fusion proteins of thisExample to positive cells is measured to be approximately 50,000-200,000MFI with a K_(D) within the range of 0.01-100 nM.

The ribosome inactivation abilities of melanoma cell-targeting fusionproteins of this Example are determined in a cell-free, in vitro proteintranslation as described above in the previous Examples. The inhibitoryeffect of the cell-targeting molecules of this Example on cell-freeprotein synthesis is significant. For certain melanoma cell-targetingfusion proteins, the ICso for protein synthesis in this cell-free assayis approximately 0.1-100 pM.

Determining the CD8+ Epitope-Peptide Cargo Delivery Functions ofMelanoma Cell-Targeting Molecules

The successful delivery of a T-cell epitope can be determined bydetecting specific cell surface, MHC class I molecule/epitope complexes(pMHC Is). In order to test whether a cell-targeting molecule candeliver CD8+ T-cell epitope cargo to the MHC class I presentationpathway of target cells, routine assays are employed which detect human,MHC Class I molecules complexed with specific epitopes, such as, e.g.one or more assays described in Examples I and 2.

Melanoma cells treated with melanoma cell-targetingfusion proteins showa positive signal for cell-surface, C1-2-epitopeMHC class I complexes,often on the majority of the treated cells depending on the incubationduration. The detection of the T-cell epitope (e.g., C1-2 (SEQ IDNO:20)) complexed with human MHC class I molecules on the cell surfaceof cell-targeting molecule treated target cells demonstrates that thecell-targeting molecule is capable of entering target melanoma cells,performing sufficient sub-cellular routing, and delivering sufficientCD8+ T-cell epitope-peptide cargo to the MHC class I pathway for surfacepresentation by target cell.

Determining the Cvtotoxicity of Melanoma Cell-Targeting Molecules Usinga Cell-Kill Assay

The cytotoxicity characteristics of melanoma cell-targeting fusionproteins of this Example are determined by the general cell-kill assayas described above in the previous Examples using melanoma cells. TheCD₅₀ values of the cell-targeting molecules of this Example areapproximately 0.01-100 nM for melanoma cells depending on the cell line.In addition, the induction of intermolecular CD8+ T-cell engagement ofmelanoma target cells presenting the delivered CD8+ T-cell epitope andcytotoxicity of melanoma cell-targeting fusion proteins of this Exampleis investigated for indirect cytotoxicity by heterologous, CD8+ T-cellepitope delivery and presentation leading to CTL-mediated cytotoxicityusing assays known to the skilled worker and/or described herein.

Determining the In Vivo Effects of the Melanoma Cell-Targeting MoleculesUsing Animal Models

Animal models are used to determine the in vivo effects of certainmelanoma cell-targeting fusion proteins of this Example on melanomacells (see e.g. Cheung M et al., Mol Cancer 9: 28 (2010)). Various micestrains are used to test the effect of intravenous administration ofmelanoma cell-targeting fusion proteins of this Example on humanmelanoma cells in mice. Cell killing effects are investigated for bothdirect cytotoxicity and indirect cytotoxicity by CD8+ T-cell epitopedelivery and presentation leading to CTL-mediated cytotoxicity usingassays known to the skilled worker and/or described herein. Optionally,“inactive” variants of the cell-targeting molecules of this Example(e.g. E167D) are used to investigate indirect cytotoxicity by CD8+T-cell epitope delivery in the absence of the catalytic activity of anyShiga toxin effector polypeptide component of the cell-targetingmolecule.

Example 4. MHC-Epitope-Complex-Targeting, Cell-Targeting MoleculesComprising Shiga Toxin A Subunit Effector Polypeptides and CD8+ T-CellEpitope-Peptides

In this Example, the Shiga toxin effector polypeptide is derived fromthe A subunit of Shiga-like Toxin 1 (SLT-A) as described above withamino acid residue substitutions conferring de-immunization (see e.g. WO2015/113005: WO 2015/113007; WO 2016/196344), CD8+ T-cellhyperimmunization (see e.g. WO 2015/113005; WO 2016/196344), andfurin-cleavage resistance, such as, e.g., R248AiR251A (WO 2015/191764;WO 2016/196344). A human, CD8+ T-cell epitope-peptide is selected basedon MHC I molecule binding predictions, HLA types, already characterizedimmunogenicities, and/or readily available reagents as described above,such as the C1-2 epitope-peptide GLDRNSGNY (SEQ ID NO:20). Aproteinaceous binding region is derived from a T-cell receptor likeantibody specific for a particular epitope-peptide-MHC molecule complex,such as, e.g., WT1 abl (US2014/294841) which is capable of specificallybinding HLA-A0201 bound to a Wilms' tumor oncogene protein (WT1)epitope-peptide RMFPNAPYL. HLA-A2-WTl complexes may be expressed byvarious cancer cell types, such as breast cancer, ovarian cancer,prostate cancer, chronic myelocytic leukemia, multiple myeloma (MM),acute lymphoblastic leukemia (ALL), acute myeloid/myelogenous leukemia(AML), and myelodysplastic syndrome (MDS) cancer cells.

Construction and Production of MHC-Epitope-Complex-Targeting,Cell-Targeting Fusion Proteins

The immunoglobulin-type binding region aHLA-WT1, the Shiga toxineffector polypeptide, and the CD8+ T-cell epitope are fused together toform a single, continuous polypeptide, such as “SLT-1A::CI-2::aHLA-WTI”or “SLT-IA::aHLA-WTI::CI-2,” and, optionally, a KDEL is added to thecarboxy terminus of the resulting polypeptide.

Determining the In Vitro Characteristics ofMHC-Epitope-Complex-Targeting, Cell-Targeting Molecules

The binding characteristics of cell-targeting molecules of this Examplefor specific MHC I molecule-epitope complex positive cells and negativecells is determined by fluorescence-based, flow-cytometry. The Bmax forcertain MHC-epitope-complex-targeting, cell-targeting fusion proteins ofthis Example to positive cells is measured to be approximately50,000-200,000 MFI with a K_(D) within the range of 0.01-100 nM, whereasthere is no significant binding to the respective MHC-epitope complexnegative cells in this assay.

The ribosome inactivation abilities of MHC-epitope-complex-targeting,cell-targeting fusion proteins of this Example are determined in acell-free, in vitro protein translation as described above in theprevious Examples. The inhibitory effect of the cell-targeting moleculesof this Example on cell-free protein synthesis is significant. Forcertain MHC-epitope-complex-targeting, cell-targeting fusion proteins,the IC₅₀ for protein synthesis in this cell-free assay is approximately0.1-100 pM.

Determining the CD8+ Epitope-Peptide Cargo Delivery Functions ofMHC-Epitope-Complex-Targeting, Cell-Targeting Molecules

The successful delivery of a T-cell epitope can be determined bydetecting specific cell surface, MHC class I molecule/epitope complexes(pMHC Is). In order to test whether a cell-targeting molecule candeliver CD8+ T-cell epitope cargo to the MHC class I presentationpathway of target cells, routine assays are employed which detect human,MHC Class I molecules complexed with specific epitopes, such as, e.g.one or more assays described in Examples 1 and 2.

Cells treated with MHC-epitope-complex-targeting, cell-targeting fusionproteins show a positive signal for cell-surface, C1-2-epitope/MHC classI complexes, often on the majority of the treated cells depending on theincubation duration. The detection of the T-cell epitope C1-2 (SEQ IDNO:20) complexed with human MHC class I molecules on the cell surface ofcell-targeting molecule treated target cells demonstrates that thecell-targeting molecule is capable of entering target cells, performingsufficient sub-cellular routing, and delivering sufficient CD8+ T-cellepitope-peptide cargo to the MHC class I pathway for surfacepresentation by target cell.

Determining the Cvtotoxicity of MHC-Epitope-Complex-Targeting,Cell-Targeting Molecules Using a Cell-Kill Assay

The cytotoxicity characteristics of MHC-epitope-complex-targeting,cell-targeting fusion proteins of this Example are determined by thegeneral cell-kill assay as described above in the previous Examplesusing specific MHC-epitope complex positive cells. In addition, theselective cytotoxicity characteristics of the sameMHC-epitope-complex-targeting, cell-targeting fusion proteins of thisExample are determined by the same general cell-kill assay using therespective MHC-epitope complex negative cells as a comparison to theappropriate MHC-epitope complex positive cells. The CD₅₀ values of thecell-targeting molecules of this Example are approximately 0.01-100 nMfor MHC-epitope complex positive cells depending on the cell line. TheCD₅₀ values of MHC-epitope-complex-targeting, cell-targeting fusionproteins of this Example are approximately 10-10,000 fold greater (lesscytotoxic) for cells not expressing the appropriate MHC-epitope complexon a cellular surface as compared to cells which do express theappropriate MHC-epitope complex on a cellular surface. In addition, theinduction of intermolecular CD8+ T-cell engagement of C1-2-presentingtarget cells and cytotoxicity of MHC-epitope-complex targeting,cell-targeting fusion proteins of this Example is investigated forindirect cytotoxicity by heterologous, CD8+ T-cell epitope delivery andpresentation leading to CTL-mediated cytotoxicity using assays known tothe skilled worker and/or described herein.

Determining the In Vivo Effects of the MHC-Epitope-Complex-Targeting,Cell-Targeting Molecules Using Animal Models

Animal models are used to determine the in viv, effects of certainMHC-epitope-complex-targeting, cell-targeting fusion proteins of thisExample on neoplastic cells. Various mice strains are used to test theeffect of intravenous administration of MHC-epitope-complex-targeting,cell-targeting fusion proteins of this Example on specific MHC-epitopecomplex positive cells in mice. Cell killing effects are investigatedfor both direct cytotoxicity and indirect cytotoxicity by CD8+ T-cellepitope delivery and presentation leading to CTL-mediated cytotoxicityusing assays known to the skilled worker and/or described herein.

Optionally, “inactive” variants of the cell-targeting molecules of thisExample (e.g. E167D) are used to investigate indirect cytotoxicity byCD8+ T-cell epitope delivery in the absence of the catalytic activity ofany Shiga toxin effector polypeptide component of the cell-targetingmolecule.

Example 5. IL-2R-targeting, Cell-Targeting Molecules Comprising ShigaToxin A Subunit Effector Polypeptides and CD8+ T-Cell Epitope-Peptides

In this Example, the Shiga toxin effector polypeptide is derived fromthe A subunit of Shiga-like Toxin 1 (SLT-1A) as described above withamino acid residue substitutions conferring de-immunization (see e.g. WO2015/113005; WO 2015/113007; WO 2016/196344), CD8+ T-cellhyperimmunization (see e.g. WO 2015/113005; WO 2016/196344), andfurin-cleavage resistance, such as, e.g., R248A/R251A (WO 2015/191764;WO 2016/196344). A human, CD8+ T-cell epitope-peptide is selected basedon MHC I molecule binding predictions, HLA types, already characterizedimmunogenicities, and/or readily available reagents as described above,such as the C1-2 epitope-peptide GLDRNSGNY (SEQ ID NO:20). Aproteinaceous binding region is derived from a ligand (the cytokineinterleukin 2 or IL-2) for the human interleukin 2 receptor (IL-2R),which is capable of specifically binding an extracellular part of thehuman IL-2R. IL-2R is a cell-surface receptor expressed by variousimmune cell types, such as T-cells and natural killer cells.

Construction and Production of IL-2R-Targeting, Cell-Targeting FusionProteins

The ligand-type binding region alIL-2R, the Shiga toxin effectorpolypeptide, and the CD8+ T-cell epitope are fused together to form asingle, continuous polypeptide, such as “C1-2::SLT-1A::IL-2” or“IL-2::C1-2::SLT-1A,” and, optionally, a KDEL is added to the carboxyterminus of the resulting polypeptide.

Determining the In Vitro Characteristics of IL-2R-Targeting,Cell-Targeting Molecules

The binding characteristics of cell-targeting molecules of this Examplefor IL-2R positive cells and IL-2R negative cells is determined byfluorescence-based, flow-cytometry. The Bmax for certainIL-2R-targeting, cell-targeting fusion proteins of this Example topositive cells is measured to be approximately 50,000-200,000 MFI with aK_(D) within the range of 0.01-100 nM, whereas there is no significantbinding to IL-2R negative cells in this assay.

The ribosome inactivation abilities of IL-2R-targeting, cell-targetingfusion proteins of this Example are determined in a cell-free, in vitroprotein translation as described above in the previous Examples. Theinhibitory effect of the cell-targeting molecules of this Example oncell-free protein synthesis is significant. For certain IL-2R-targeting,cell-targeting fusion proteins, the ICso for protein synthesis in thiscell-free assay is approximately 0.1-100 pM.

Determining the CD8+ Epitope-Peptide Cargo Delivery Functions ofIL-2R-Targeting, Cell-Targeting Molecules

The successful delivery of a T-cell epitope can be determined bydetecting specific cell surface, MHC class I molecule/epitope complexes(pMHC Is). In order to test whether a cell-targeting molecule candeliver CD8+ T-cell epitope cargo to the MHC class I presentationpathway of target cells, routine assays are employed which detect human,MHC Class I molecules complexed with specific epitopes, such as, e.g.one or more assays described in Examples 1 and 2.

Cells treated with IL-2R-targeting, cell-targeting fusion proteins showa positive signal for cell-surface, C1-2-epitope/MHC class I complexes,often on the majority of the treated cells depending on the incubationduration. The detection of the T-cell epitope C1-2 (SEQ ID NO:20)complexed with human MHC class I molecules on the cell surface ofcell-targeting molecule treated target cells demonstrates that thecell-targeting molecule is capable of entering target cells, performingsufficient sub-cellular routing, and delivering sufficient CD8+ T-cellepitope-peptide cargo to the MHC class I pathway for surfacepresentation by target cell.

Determining the Cytotoxicity of IL-2R-Targeting, Cell-TargetingMolecules Using a Cell-Kill Assay

The cytotoxicity characteristics of IL-2R-targeting, cell-targetingfusion proteins of this Example are determined by the general cell-killassay as described above in the previous Examples using IL-2R positivecells. In addition, the selective cytotoxicity characteristics of thesame IL-2R-targeting, cell-targeting fusion proteins of this Example aredetermined by the same general cell-kill assay using IL-2R negativecells as a comparison to the IL-2R positive cells. The CD₅₀ values ofthe cell-targeting molecules of this Example are approximately 0.01-100nM for IL-2R positive cells depending on the cell line. The CD₅₀ valuesof IL-2R-targeting, cell-targeting fusion proteins of this Example areapproximately 10-10,000 fold greater (less cytotoxic) for cells notexpressing IL-2R on a cellular surface as compared to cells which doexpress IL-2R on a cellular surface. In addition, the induction ofintermolecular CD8+ T-cell engagement of C1-2-presenting target cellsand cytotoxicity of IL-2R-targeting, cell-targeting fusion proteins ofthis Example is investigated for indirect cytotoxicity by heterologous,CD8+ T-cell epitope delivery and presentation leading to CTL-mediatedcytotoxicity using assays known to the skilled worker and/or describedherein.

Determining the In Vivo Effects of the IL-2R-Targeting, Cell-TargetingMolecules Using Animal Models

Animal models are used to determine the in vivo effects of certainIL-2R-targeting, cell-targeting fusion proteins of this Example onneoplastic cells. Various mice strains are used to test the effect ofintravenous administration of IL-2R-targeting, cell-targeting fusionproteins of this Example on IL-2R positive cells in mice. Cell killingeffects are investigated for both direct cytotoxicity and indirectcytotoxicity by CD8+ T-cell epitope delivery and presentation leading toCTL-mediated cytotoxicity using assays known to the skilled workerand/or described herein. Optionally, “inactive” variants of thecell-targeting molecules of this Example (e.g. E167D) are used toinvestigate indirect cytotoxicity by CD8+ T-cell epitope delivery in theabsence of the catalytic activity of any Shiga toxin effectorpolypeptide component of the cell-targeting molecule.

Example 6. CEA-targeting, Cell-Targeting Molecules Comprising a ShigaToxin Effector Polypeptide and a Heterologous, CD8+ T-Cell Epitope

Carcinoembryonic antigens (CEAs) expression in adult humans isassociated with cancer cells, such as, e.g., adenocarcinomas of thebreast, colon, lung, pancreas, and stomach. In this example, the Shigatoxin effector polypeptide is derived from the A subunit of Shiga Toxin(StxA) (SEQ ID NO:2) as described above with amino acid residuesubstitutions conferring de-immunization (see e.g. WO 2015/113005; WO2015/113007: WO 2016/196344), CD8+ T-cell hyperimmunization (see e.g. WO2015/113005: WO 2016/196344), and furin-cleavage resistance, such as,e.g., R248A/R251A (WO 2015/191764; WO 2016/196344). A human, CD8+ T-cellepitope-peptide is selected based on MHC I molecule binding predictions,HLA types, already characterized immunogenicities, and/or readilyavailable reagents as described above, such as the F3-epitope ILRGSVAHK(SEQ ID NO:26) described in WO 2015/113005 and WO 2016/196344. Theimmunoglobulin-type, binding region aCEA, which binds specifically andwith high-affinity to an extracellular antigen on human carcinoembryonicantigen (CEA), such as the tenth human fibronectin type III domainderived binding region C743 as described in Pirie C et al., J Biol Chem286: 4165-72 (2011).

Construction, Production, and Purification of CEA-targeting,Cell-Targeting Molecules

The Shiga toxin effector polypeptide, aCEA binding region polypeptide,and heterologous, CD8+ T-cell epitope-peptide are operably linkedtogether using standard methods known to the skilled worker to formcell-targeting molecules of the present invention. For example, fusionproteins are produced by expressing a polynucleotide encoding one ormore of StxA::aCEA::F3, StxA::F3::aCEA, aCEA::StxA::F3, F3::aCEA::StxA,aCEA::F3::StxA, and F3::StxA::atCEA, which each optionally have one ormore proteinaceous linkers described herein between the fusedproteinaceous components. Expression of these exemplary CEA-targetingfusion proteins is accomplished using either bacterial and/or cell-free,protein translation systems as described in the previous Examples.

Determining the In Vitro Characteristics ofExemplary CEA-Targeting,Cell-Targeting Fusion Proteins

The binding characteristics of cell-targeting molecule of this Examplefor CEA positive cells and CEA negative cells is determined byfluorescence-based, flow-cytometry. The B, for StxA::aCEA::F3,StxA::F3::aCEA, aCEA::StxA::F3, F3::aCEA::StxA, aCEA::F3::StxA, andF3::StxA::aCEA to CEA positive cells are each measured to beapproximately 50,000-200,000 MFI with a K_(D) within the range of0.01-100 nM, whereas there is no significant binding to CEA negativecells in this assay.

The ribosome inactivation abilities of the fusion proteins of thisExample are determined in a cell-free, in vitro protein translation asdescribed above in the previous Examples. The inhibitory effect of thecytotoxic fusion proteins of this Example on cell-free protein synthesisare significant. The ICso values on protein synthesis in this cell-freeassay measured for StxA::αCEA::F3, StxA::F3::αCEA, αCEA::StxA::F3,F3::αCEA::StxA, αCEA::F3::StxA, and F3::StxA::αCEA are eachapproximately 0.1-100 pM.

Determining the CD8+ Epitope-Peptide Cargo Delivery Functions ofCEA-Targeting, Cell-Targeting Molecules

The successful delivery of a T-cell epitope can be determined bydetecting specific cell surface, MHC class I molecule/epitope complexes(pMHC Is). In order to test whether a cell-targeting molecule candeliver CD8+ T-cell epitope cargo to the MHC class I presentationpathway of target cells, routine assays are employed which detect human,MHC Class I molecules complexed with specific epitopes, such as, e.g.one or more assays described in Examples 1 and 2.

Cells treated with CEA-targeting, cell-targeting fusion proteins show apositive signal for cell-surface, F3-epitope/MHC class I complexes,often on the majority of the treated cells depending on the incubationduration. The detection of the CD8+ T-cell epitope F3 (SEQ ID NO:26)complexed with human MHC class I molecules on the cell surface ofcell-targeting molecule treated target cells demonstrates that thecell-targeting molecule is capable of entering target cells, performingsufficient sub-cellular routing, and delivering sufficient CD8+ T-cellepitope-peptide cargo to the MHC class I pathway for surfacepresentation by target cell.

Determining the Cytotoxicity of Exemplary CEA-Targeting, Cell-TargetingFusion Proteins Using a Cell-Kill Assay

The cytotoxicity characteristics of cell-targeting molecule of thisExample are determined by the general cell-kill assay as described abovein the previous Examples using CEA positive cells. In addition, theselective cytotoxicity characteristics of the exemplary CEA-targeting,cell-targeting fusion proteins are determined by the same generalcell-kill assay using CEA negative cells as a comparison to the CEAantigen positive cells. The CD₅₀ values measured for StxA::αCEA:tF3,StxA::F3::αCEA, αCEA::StxA::F3, F3::αCEA::StxA, αCEA::F3::StxA, andF3::StxA::αCEA are approximately 0.01-100 nM for CEA positive cellsdepending on the cell line. The CD₅₀ values of the CEA-targeting,cell-targeting fusion proteins of this Example are approximately10-10,000 fold greater (less cytotoxic) for cells not expressing CEA ona cellular surface as compared to cells which do express CEA on acellular surface. In addition, the induction of intermolecular CD8+T-cell engagement of F3-presenting target cells and cytotoxicity ofStxA::αCEA::F3, StxA::F3::αCEA, αCEA::StxA::F3, F3::αCEA::StxA,αCEA::F3::StxA, and F3::StxA::αCEA is investigated for indirectcytotoxicity by heterologous, CD8+ T-cell epitope delivery andpresentation leading to CTL-mediated cytotoxicity using assays known tothe skilled worker and/or described herein.

Determining the In Vimv Effects of an Exemplary CEA-Targeting,Cell-Targeting Fusion Protein Using Animal Models

Animal models are used to determine the in vivo effects exemplaryCEA-targeting fusion proteins on neoplastic cells. Various mice strainsare used to test the effects on xenograft tumors of the cell-targetingfusion proteins StxA::αCEA::F3. StxA::F3::αCEA, αCEA::StxA::F3,F3::αCEA::StxA, αCEA::F3::StxA, and F3::StxA::αCEA after intravenousadministration to mice injected with human neoplastic cells whichexpress CEA(s) on their cell surfaces. Cell killing is investigated forboth direct cytotoxicity and indirect cytotoxicity by CD8+ T-cellepitope cargo delivery and presentation leading to CTL-mediatedcytotoxicity using assays known to the skilled worker and/or describedherein. Optionally, “inactive” variants of the cell-targeting moleculesof this Example (e.g. E167D) are used to investigate indirectcytotoxicity caused by CD8+ T-cell epitope delivery in the absence ofthe catalytic activity of any Shiga toxin effector polypeptide componentof the cell-targeting molecule.

Example 7. HER2-targeting, Cell-Targeting Molecules Comprising a ShigaToxin Effector Polypeptide and a Heterologous, CD8+ T-Cell Epitope

HER2 overexpression has been observed in breast, colorectal,endometrial, esophageal, gastric, head and neck, lung, ovarian,prostate, pancreatic, and testicular germ cell tumor cells. In thisExample, the Shiga toxin effector polypeptide is derived from the ASubunit of StxA (SEQ ID NO:2) as described above with amino acid residuesubstitutions conferring de-immunization (see e.g. WO 2015/113005; WO2015/113007; WO 2016/196344), CD8+ T-cell hyperimmunization (see e.g. WO2015/113005; WO 2016/196344), and furin-cleavage resistance, such as,e.g., R248A/R251A (WO 2015/191764; WO 2016/196344). A human, CD8+ T-cellepitope-peptide is selected based on MHC I molecule binding predictions,HLA types, already characterized immunogenicities, and/or readilyavailable reagents as described above, such as the C3-epitope GVMTRGRLK(SEQ ID NO:22) described in WO 2015/113005 and WO 2016/196344. Thebinding region aHER2, which binds an extracellular part of human HER2,is generated by screening or selected from available immunoglobulin-typepolypeptides known to the skilled worker (see e.g. the ankyrin repeatDARPin™ G3 which binds with high affinity to an extracellular epitope ofHER2 (Goldstein R et al., Eur J Nucl Med Mol Imaging 42: 288-301(2015))).

Construction, Production, and Purification of HER2-Targeting,Cell-Targeting Molecules

The Shiga toxin effector polypeptide, aHER2 binding region polypeptide,and heterologous, CD8+ T-cell epitope-peptide are operably linkedtogether using standard methods known to the skilled worker to formcell-targeting molecules of the present invention. For example, fusionproteins are produced by expressing a polynucleotide encoding one ormore of StxA::aHER2::C3, StxA::C3::aHER2, aHER2::StxA::C3,C3::aHER2::StxA, aHER2::C3::StxA, and C3::StxA::aHER2, which eachoptionally have one or more proteinaceous linkers described hereinbetween the fused proteinaceous components. Expression of theseexemplary HER2-targeting fusion proteins is accomplished using eitherbacterial and/or cell-free, protein translation systems as described inthe previous Examples.

Determining the In Vitro Characteristics of Exemplary HER2-Targeting,Cell-Targeting Fusion Proteins

The binding characteristics of cell-targeting molecule of this Examplefor HER2 positive cells and HER2 negative cells is determined byfluorescence-based, flow-cytometry. The B_(max) for StxA::αHER2::C3,StxA::C3::αHER2, αHER2::StxA::C3, C3::αHER2::StxA, αHER2::C3::StxA, andC3::StxA::αHER2 to HER2 positive cells are each measured to beapproximately 50,000-200,000 MFI with a K_(D) within the range of0.01-100 nM, whereas there is no significant binding to HER2 negativecells in this assay.

The ribosome inactivation abilities of the fusion proteins of thisExample are determined in a cell-free, in vitro protein translation asdescribed above in the previous Examples. The inhibitory effect of thecytotoxic fusion proteins of this Example on cell-free protein synthesisare significant. The ICso values on protein synthesis in this cell-freeassay measured for StxA::αHER2::C3. StxA::C3::αHER2, αHER2::StxA::C3.C3::αHER2::StxA, αHER2::C3::StxA, and C3::StxA::αHER2 are eachapproximately 0.1-100 pM.

Determining the CD8+ Epitope-Peptide Cargo Delivery Functions ofHER2-Targeting, Cell-Targeting Molecules

The successful delivery of a T-cell epitope can be determined bydetecting specific cell surface, MHC class I molecule/epitope complexes(pMHC Is). In order to test whether a cell-targeting molecule candeliver CD8+ T-cell epitope cargo to the MHC class I presentationpathway of target cells, routine assays are employed which detect human,MHC Class I molecules complexed with specific epitopes, such as, e.g.one or more assays described in Examples 1 and 2.

Cells treated with HER2-targeting, cell-targeting fusion proteins show apositive signal for cell-surface, C3-epitope/MHC class I complexes,often on the majority of the treated cells depending on the incubationduration. The detection of the CD8+ T-cell epitope C3 (SEQ ID NO:22)complexed with human MHC class I molecules on the cell surface ofcell-targeting molecule treated target cells demonstrates that thecell-targeting molecule is capable of entering target cells, performingsufficient sub-cellular routing, and delivering sufficient CD8+ T-cellepitope-peptide cargo to the MHC class I pathway for surfacepresentation by target cell.

Determining the Cytotoxicity of Exemplary HER2-Targeting, Cell-TargetingFusion Proteins Using a Cell-Kill Assay

The cytotoxicity characteristics of cell-targeting molecule of thisExample are determined by the general cell-kill assay as described abovein the previous Examples using HER2 positive cells. In addition, theselective cytotoxicity characteristics of the exemplary HER2-targeting,cell-targeting fusion proteins are determined by the same generalcell-kill assay using HER2 negative cells as a comparison to the HER2antigen positive cells. The CD₅₀ values measured for StxA::αHER2::C3,StxA::C3::αHER2, αHER2::StxA::C3, C3::αHER2::StxA, αHER2::C3::StxA, andC3::StxA::αHER2 are approximately 0.01-100 nM for HER2 positive cellsdepending on the cell line. The CD₅₀ values of the HER2-targeting,cell-targeting fusion proteins of this Example are approximately10-10.000 fold greater (less cytotoxic) for cells not expressing HER2 ona cellular surface as compared to cells which do express HER2 on acellular surface. In addition, the induction of intermolecular CD8+T-cell engagement of C3-presenting target cells and cytotoxicity ofStxA::αHER2::C3, StxA::C3::αHER2, αHER2::StxA::C3, C3::αHER2::StxA,αHER2::C3::StxA, and C3::StxA::αHER2 is investigated for indirectcytotoxicity by heterologous, CD8+ T-cell epitope delivery andpresentation leading to CTL-mediated cytotoxicity using assays known tothe skilled worker and/or described herein.

Determining the In Vivo Effects of an Exemplary HER2-Targeting,Cell-Targeting Fusion Protein Using Animal Models

Animal models are used to determine the in vivo effects exemplaryHER2-targeting fusion proteins on neoplastic cells. Various mice strainsare used to test the effects on xenograft tumors of the cell-targetingfusion proteins StxA::αHER2::C3, StxA::C3::αHER2, αHER2::StxA::C3.C3::αHER2::StxA, αHER2::C3::StxA, and C3::StxA::αHER2 after intravenousadministration to mice injected with human neoplastic cells whichexpress HER2(s) on their cell surfaces. Cell killing is investigated forboth direct cytotoxicity and indirect cytotoxicity by CD8+ T-cellepitope cargo delivery and presentation leading to CTL-mediatedcytotoxicity using assays known to the skilled worker and/or describedherein. Optionally, “inactive” variants of the cell-targeting moleculesof this Example (e.g. E167D) are used to investigate indirectcytotoxicity caused by CD8+ T-cell epitope delivery in the absence ofthe catalytic activity of any Shiga toxin effector polypeptide componentof the cell-targeting molecule.

Example 8. Cell-Targeting Molecules Targeting Various Cell-Types, EachComprising a Shiga Toxin A Subunit Effector Polypeptide and One or More,Heterologous, CD8+ T-Cell Epitope-Peptides Located Carboxy-Terminal tothe Shiga Toxin A Subunit Effector Polypeptide Component

In this Example, three proteinaceous structures are associated with eachother to form exemplary, cell-targeting molecules of the presentinvention. In this Example, the Shiga toxin effector polypeptide isderived from the A Subunit of SLT-1A (SEQ ID NO:1) as described abovewith amino acid residue substitutions conferring de-immunization (seee.g. WO 2015/113005; WO 2015/113007; WO 2016/196344), CD8+ T-cellhyperimmunization (see e.g. WO 2015/113005; WO 2016/196344), andfurin-cleavage resistance, such as, e.g., R248A, R251A (WO 2015/191764;WO 2016/196344) One or more CD8+ T-cell epitope-peptides are selected,such as, e.g., based on MHC I molecule binding predictions, HLA types,already characterized immunogenicities, and/or readily availablereagents as described herein. A binding region component is derived fromthe immunoglobulin domain from the molecule chosen from column 1 ofTable 10 and which binds the extracellular target biomolecule indicatedin column 2 of Table 10.

Using reagents and techniques known in the art, the three components: 1)the ligand or immunoglobulin-derived binding region, 2) the Shiga toxineffector polypeptide, and 3) the CD8+ T-cell epitope-peptide(s) or alarger polypeptide comprising at least one heterologous CD8+ T-cellepitope-peptide, are associated with each other to form a cell-targetingmolecule of the present invention and, optionally, the CD8+ T-cellepitope-peptide is located carboxy-terminal to the carboxy terminus ofthe Shiga toxin A1 fragment region of the Shiga toxin effectorpolypeptide which optionally comprises a disrupted furin-cleavage motif.The exemplary cell-targeting molecules of this Example are tested asdescribed in the previous Examples using cells expressing theappropriate extracellular target biomolecules. The exemplarycell-targeting molecules of this Example may be used, e.g., to diagnoseand treat diseases, conditions, and/or disorders indicated in column 3of Table 10.

TABLE 10 Various Binding Regions for Cell Targeting Source ofExtracellular binding region target Application(s) alemtuzumab CD52B-cell cancers, such as lymphoma and leukemia, and B-cell related immunedisorders, such as autoimmune disorders basiliximab CD25 T-celldisorders, such as prevention of organ transplant rejections, and someB-cell lineage cancers brentuximab CD30 hematological cancers, B-cellrelated immune disorders, and T-cell related immune disorderscatumaxomab EpCAM various cancers, such as ovarian cancer, malignantascites, gastric cancer cetuximab EGFR various cancers, such ascolorectal cancer and head and neck cancer daclizumab CD25 B-celllineage cancers and T-cell disorders, such as rejection of organtransplants daratumumab CD38 hematological cancers, B-cell relatedimmune disorders, and T-cell related immune disorders dinutuximabganglioside Various cancers, such as breast cancer, myeloid GD2 cancers,and neuroblastoma efalizumab LFA-1 autoimmune disorders, such aspsoriasis (CD11a) ertumaxomab HER2/neu various cancers and tumors, suchas breast cancer and colorectal cancer gemtuzumab CD33 myeloid cancer orimmune disorder ibritumomab CD20 B-cell cancers, such as lymphoma andleukemia, and B-cell related immune disorders, such as autoimmunedisorders inotuzumab CD22 B-cell cancers, such as lymphoma and leukemia,and B-cell related immune disorders, such as autoimmune disordersipilimumab CD152 T-cell related disorders and various cancers, such asleukemia, melanoma muromonab CD3 prevention of organ transplantrejections natalizumab initegrin α4 autoimmune disorders, such asmultiple sclerosis and Crohn's disease obinutuzumab CD20 B-cell cancers,such as lymphoma and leukemia, and B-cell related immune disorders, suchas autoimmune disorders ocaratuzumab CD20 B-cell cancers, such aslymphoma and leukemia, and B-cell related immune disorders, such asautoimmune disorders ocrelizumab CD20 B-cell cancers, such as lymphomaand leukemia, and B-cell related immune disorders, such as autoimmunedisorders ofatumumab CD20 B-cell cancers, such as lymphoma and leukemia,and B-cell related immune disorders, such as autoimmune disorderspalivizumab F protein of treat respiratory syncytial virus respiratorysyncytial virus panitumumab EGFR various cancers, such as colorectalcancer and head and neck cancer pertuzumab HER2/neu various cancers andtumors, such as breast cancer and colorectal cancer pro 140 CCR5 HIVinfection and T-cell disorders ramucirumab VEGFR2 various cancers andcancer related disorders, such as solid tumors rituximab CD20 B-cellcancers, such as lymphoma and leukemia, and B-cell related immunedisorders, such as autoimmune disorders tocilizumab or IL-6 receptorautoimmune disorders, such as rheumatoid atlizumab arthritis tositumomabCD20 B-cell cancers, such as lymphoma and leukemia, and B-cell relatedimmune disorders, such as autoimmune disorders trastuzumab HER2/neuvarious cancers and tumors, such as breast cancer and colorectal cancerublituximab CD20 B-cell cancers, such as lymphoma and leukemia, andB-cell related immune disorders, such as autoimmune disordersvedolizumab integrin α4β7 autoimmune disorders, such as Crohn's diseaseand ulcerative colitis CD20 binding scFv(s) CD20 B-cell cancers, such aslymphoma and leukemia, Geng S et al., Cell and B-cell related immunedisorders, such as Mol Immunol 3: 439- autoimmune disorders 43 (2006);Olafesn T et al., Protein Eng Des Sel 23: 243-9 (2010) CD22 bindingscFv(s) CD22 B-cell cancers or B-cell related immune disorders Kawas Set al., MAbs 3: 479-86 (2011) CD25 binding scFv(s) CD25 various cancersof the B-cell lineage and immune Muramatsu H et al., disorders relatedto T-cells Cancer Lett 225: 225- 36 (2005) CD30 binding CD30 B-cellcancers or B-cell/T-cell related immune monoclonal antibody(s) disordersKlimka A et al., Br J Cancer 83: 252-60 (2000) CD33 binding CD33 myeloidcancer or immune disorder monoclonal antibody(s) Benedict C et al., JImmunol Methods 201: 223-31 (1997) CD38 binding CD38 hematologicalcancers, B-cell related immune immunoglobulin disorders, and T-cellrelated immune disorders domains U.S. Pat. No. 8,153,765 CD40 bindingscFv(s) CD40 various cancers and immune disorders Ellmark P et al.,Immunology 106: 456- 63 (2002) CD52 binding CD52 B-cell cancers, such aslymphoma and leukemia, monoclonal and B-cell related immune disorders,such as antibody(s) autoimmune disorders U.S. Pat. No. 7,910,104 B2 CD56binding CD56 immune disorders and various cancers, such as monoclonalantibody(s) lung cancer, Merkel cell carcinoma, myeloma Shin J et al.,Hybridoma 18: 521-7 (1999) CD79 binding CD79 B-cell cancers or B-cellrelated immune disorders monoclonal antibody(s) Zhang L et al., TherImmunol 2: 191-202 (1995) CD248 binding CD248 various cancers, such asinhibiting angiogenesis scFv(s) Zhao A et al., J Immunol Methods 363:221-32 (2011) EpCAM binding EpCAM various cancers, such as ovariancancer, monoclonal antibody(s) malignant ascites, gastric cancerSchanzer J et al., J Immunother 29: 477- 88 (2006) PSMA binding PSMAprostate cancer monoclonal antibody(s) Frigerio B et al., Eur J Cancer49: 2223-32 (2013) Eph-B2 binding Eph-B2 for various cancers such ascolorectal cancer and monoclonal antibody(s) prostate cancer Abéngozar Met al., Blood 119: 4565-76 (2012) Endoglin binding Endoglin variouscancers, such as breast cancer and monoclonal antibody(s) colorectalcancers Völkel T et al., Biochim Biophys Res Acta 1663: 158-66 (2004)FAP binding FAP various cancers, such as sarcomas and bone monoclonalantibody(s) cancers Zhang J et al., FASEB J 27: 581-9 (2013) CEA bindingCEA various cancers, such as gastrointestinal cancer, antibody(s) andpancreatic cancer, lung cancer, and breast cancer scFv(s) Neumaier M etal., Cancer Res 50: 2128-34 (1990); Pavoni E et al., BMC Cancer 6: 4(2006); Yazaki P et al., Nucl Med Biol 35: 151-8 (2008); Zhao J et al.,Oncol Res 17: 217-22 (2008) CD24 binding CD24 various cancers, such asbladder cancer monoclonal anlibody(s) Kristiansen G et al., Lab Invest90: 1102- 16 (2010) LewisY antigen LewisY various cancers, such ascervical cancer and binding scFv(s) antigens uterine cancer Power B etal., Protein Sci 12: 734-47 (2003); monoclonal antibody BR96 Feridani Aet al., Cytometry 71: 361-70 (2007) adalimumab TNF-α various cancers andimmune disorders, such as Rheumatoid arthritis, Crohn's Disease, PlaquePsoriasis, Psoriatic Arthritis, Ankylosing Spondylitis, JuvenileIdiopathic Arthritis, Hemolytic disease of the newborn afelimomab TNF-αvarious cancers and immune disorders ald518 IL-6 various cancers andimmune disorders, such as rheumatoid arthritis anrukinzumab or ima-IL-13 various cancers and immune disorders 638 briakinumab IL-12, IL-23various cancers and immune disorders, such as psoriasis, rheumatoidarthritis, inflammatory bowel diseases, multiple sclerosis brodalumabIL-17 various cancers and immune disorders, such as inflammatorydiseases canakinumab IL-1 various cancers and immune disorders, such asrheumatoid arthritis certolizumab TNF-α various cancers and immunedisorders, such as Crohn's disease fezakinumab IL-22 various cancers andimmune disorders, such as rheumatoid arthritis, psoriasis ganitumabIGF-I various cancers golimumab TNF-α various cancers and immunedisorders, such as rheumatoid arthritis, psoriatic arthritis, ankylosingspondylitis infliximab TNF-α various cancers and immune disorders, suchas rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis,psoriasis, Crohn's disease, ulcerative colitis ixekizumab TL-17A variouscancers and immune disorders, such as autoimmune diseases mepolizumabIL-5 various immune disorders and cancers, such as B-cell cancersnerelimomab TNF-α various cancers and immune disorders olokizumab IL6various cancers and immune disorders ozoralizumab TNF-α inflammationperakizumab IL17A various cancers and immune disorders, such asarthritis placulumab human TNF various immune disorders and cancerssarilumab IL6 various cancers and immune disorders, such as rheumatoidarthritis, ankylosing spondylitis siltuximab IL-6 various cancers andimmune disorders sirukumab IL-6 various cancers and immune disorders,such as rheumatoid arthritis tabalumab BAFF B-cell cancers ticilimumabor CTLA-4 various cancers tremelimumab tildrakizumab IL23immunologically mediated inflammatory disorders tnx-650 IL-13 variouscancers and immune disorders, such as B-cell cancers tocilizumab or IL-6receptor various cancers and immune disorders, such as atlizumabrheumatoid arthritis ustekinumab IL-12, IL-23 various cancers and immunedisorders, such as multiple sclerosis, psoriasis, psoriatic arthritisVarious growth VEGFR, various cancer, such as breast cancer and colonfactors: VEGF, EGF1, EGFR, FGFR cancer, and to inhibit vascularizationEGF2, FGF Various cytokines: IL-2, IL-2R, IL-6R, various immunedisorders and cancers IL-6, IL-23, CCL2, IL-23R, BAFFs, TNFs, CD80/CD86,RANKL TNFRSF13/ TNFRSF17, TNFR Broadly neutralizing Influenza viralinfections antibodies identified surface from patient samples antigens,e.g. Prabakaran et al., hemagglutinins Front Microbiol 3: and influenza277 (2012) matrix protein 2 Broadly neutralizing Coronavirus viralinfections antibodies identified surface from patient samples antigensPrabakaran et al., Front Microbiol 3: 277 (2012) Broadly neutralizingHenipaviruses viral infections antibodies identified surface frompatient samples antigens Prabakaran et al., Front Microbiol 3: 277(2012)

While some embodiments of the invention have been described by way ofillustration, it will be apparent that the invention may be put intopractice with many modifications, variations and adaptations, and withthe use of numerous equivalents or alternative solutions that are withinthe scope of persons skilled in the art, without departing from thespirit of the invention or exceeding the scope of the claims.

All publications, patents, and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety. The international patent application publications WO2014/164693, WO 2014/164680, WO 2015/138435, WO 2015/138452, WO2015/113005, WO 2015/113007, WO 2015/191764, WO 2016/196344, and WO2017/019623, are each incorporated herein by reference in its entirety.The disclosures of U.S. patent application publications US 2007/298434,US 2009/156417, US 2013/196928, US 2016/177284, US 2017/143814, and US2017/275382 are each incorporated here by reference in their entirety.The disclosure of WO 2018/106895 is incorporated herein by reference inits entirety. The complete disclosures of all electronically availablebiological sequence information from GenBank (National Center forBiotechnology Information, U.S.) for amino acid and nucleotide sequencescited herein are each incorporated herein by reference in their entirety

Sequence Listing ID Text Number Description Biological Sequence SEQ IDShiga-like KEFFLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDN NO: 1toxin 1 LFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAD Subunit AFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLD (SLT-1A)LMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARMASDEFPSMCPADGRVRGITHNKILWDS STLGAILMRRTISS SEQ IDShiga toxin KEFFLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDN NO: 2Subunit A LFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAD (StxA)FSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARMASDEFPSMCPADGRVRGITHNKILWDS STLGAILMRRTISS SEQ IDShiga-like DEFTVDFSSQKSYVDSLNSIRSAISTPLGNISQGGVSVSVINHVLGGN NO: 3toxin 2 YISLNVRGLDPYSERFNHLRLIMERNNLYVAGFINTETNIFYRFSDFS Subunit AHISVPDVITVSMTTDSSYSSLQRIADLERTGMQIGRHSLVGSYLDLM (SLT-2A)EFRGRSMTRASSRAMLRFVTVIAEALRFRQIQRGFRPALSEASPLYTMTAQDVDLTLNWGRISNVLPEYRGEEGVRIGRISFNSLSAILGSVAVILNCHSTGSYSVRSVSQKQKTECQIVGDRAAIKVNNVLWEANTIAA LLNRKPQDLTEPNQ SEQ IDShiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGTGDN NO: 4subtype c LFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAD Subunit AFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLD (Stx1cA)LMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSVNAIL GSVALILNCHHHASRVAR SEQ IDShiga toxin KEFTLDFSTAKKYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGTGDN NO: 5subtype d LFAVDIMGLEPEEERFNNLRLIVERNNLYVTGFVNRTNNVFYRFAD Subunit AFSHVTFPGTRAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLD (Stx1dA)LMSYSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSILPDYHGQDSVRVGRISFGSINAILG SVALILNCHHHASRVAR SEQ IDShiga toxin QDFTVDFSTAKKYVDSLNAIRSAIGTPLHSISSGGTSLLMIDNGTGD NO: 6subtype e NLFAVDIRGLDPEEERFDNLRLIIERNNLYVTGFVNRTSNIFYRFADF Subunit ASHVTFPGTRAVTLSGDSSYTTLQRVAGIGRTGMQINRHSLTTSYLDL (Stx1eA)MSYSGSSLTQPVARAMLRFVTVTAEALRFRQIQRGFRTTLDDVSGHSYTMTVEDVDLTLNWGRLSSVLPDYHGDSVRVGRISFGGVNAILG SVALILNCHHHTSRVSR SEQ IDShiga toxin REFTIDFSTQQSYVSSLNSIRTEISTPLEHISQGTTSVSVINHTPPGSYF NO: 7subtype 2c AVDIRGLDVYQARFDHLRLIIEQNNLYVAGFVNTATNTFYRFSDFT Subunit AHISVPGVTTVSMTTDSSYTTLQRVAALERSGMQISRHSLVSSYLALM (Stx2cA)EFSGNTMTRDASRAVLRFVTVTAEALRFRQIQREFRQALSETAPVY variant 1TMTPGDVDLTLNWGRISNVLPEYRGEDGYRVGRISFNNISAILGTVA VILNCHHQGARSVR SEQ IDShiga toxin REFTIDFSTQQSYVSSLNSIRTEISTPLEHISQGTTSVSVINHTPPGSYF NO: 8subtype 2c AVDIRGLDVYQARFDHLRLIIEQNNLYVAGFVNTATNTFYRFSDFT Subunit AHISVPGVTTVSMTTDSSYTTLQRVAALERSGMQISRHSLVSSYLALM (Stx2cA)EFSGNTMTRDASRAVLRFVTVTAEALRFRQIQREFRQALSETAPVY variant 2TMTPGDVDLTLNWGRISNVLPEYRGEDGYRVGRISFNNISAILGTVA VILNCHHQGARSVR SEQ IDShiga toxin REFTIDFSTQQSYVSSLNSIRTEISTPLEHISQGTTSVSVINHTPPGSYF NO: 9subtype 2c AVDIRGLDIYQARFDHLRLIIEQNNLYVAGFVNTATNTFYRFSDFTHI Subunit ASVPGVTTVSMTTDSSYTTLQRVAALERSGMQISRHSLVSSYLALME (Stx2cA)FSGNTMTRDASRAVLRFVTVTAEALRFRQIQREFRQALSETAPVYT variant 3MTPGDVDLTLNWGRISNVEPEYRGEDGVRVGRISFNNISAILGTVA VILNCHHQGARSVR SEQ IDShiga toxin REFTIDFSTQQSYVSSLNSIRTEISTPLEHISQGTTSVSVINHTPPGSYF NO: 10subtype 2c AVDIRGLDVYQARFDHLRLIIEQNNLYVAGFVNTATNTFYRFSDFT Subunit AHISVPGVTTVSMTTDSSYTTLQRVAALERSGMQISRHSLVSSYLALM (Stx2cA)EFSGNTMTRDASRAVLRFVTVTAEALRFRQIQREFRQALSETAPVY variant 4TMTPGDVDLTLNWGRISNVLPEYRGEDGYRVGRISFNNISAILGTVA VILNCHHQGARSVR SEQ IDShiga toxin REFTIDFSTQQSYVSSLNSIRTEISTPLEHISQGTTSVSVINHTPPGSYF NO: 11subtype 2c AVDIRGLDVYQARFDHLRLIIEQNNLYVAGFVNTATNTFYRFSDFT Subunit AHISVPGVTTVSMTTDSSYTTLQRVAALERSGMQISRHSLVSSYLALM (Stx2cA)EFSGNTMTRDASRAVLRFVTVTAEALRFRQIQREFRQALSETAPVY variant 5TMTPGDVDLTLNWGRISNVLPEYRGEDGYRVGRISFNNISAILGTVA VILNCHHQGARSVR SEQ IDShiga toxin REFTIDFSTQQSYVSSLNSIRTEISTPLEHISQGTTSVSVINHTPPGSYF NO: 12subtype 2c AVDIRGLDVYQARFDHLRLIIEQNNLYVAGFVNTATNTFYRFSDFT Subunit AHISVPGVTTVSMTTDSSYTTLQRVAALERSGMQISRHSLVSSYLALM (Stx2cA)EFSGNTMTRDASRAVLRFVTVTAEALRFRQIQREFRQALSETAPVY variant 6TMTPGDVDLTLNWGRISNVLPEYRGEDGYRVGRISFNNISAILGTVA VILNCHHQGARSVR SEQ IDShiga toxin REFTIDFSTQQSYVSSLNSIRTEISTPLEHISQGTTSVSVINHTPPGSYF NO: 13subtype 2d AVDIRGLDVYQARFDHLRLIIEQNNLYVAGFVNTATNTFYRFSDFA Subunit AHISVPGVTTVSMTTDSSYTTLQRVAALERSGMQISRHSLVSSYLALM (Stx2dA)EFSGNTMTRDASRAVLRFVTVTAEALRFRQIQREFRQALSETAPVY variant 1TMTPGDVDLTLNWGRISNVLPEYRGEDGYRVGRISFNNISAILGTVA VILNCHHQGARSVR SEQ IDShiga toxin REFTIDFSTQQSYVSSLNSIRTEISTPLEHISQGTTSVSVINHTPPGSYF NO: 14subtype 2d AVDIRGLDVYQARFDHLRLIIEQNNLYVAGFVNTATNTFYRFSDFT Subunit AHISVPGVTTVSMTTDSSYTTLQRVAALERSGMQISRHSLVSSYLALM (Stx2dA)EFSGNTMTRDASRAVLRFVTVTAEALRFRQIQREFRQALSETAPVY variant 2TMTPGDVDLTLNWGRISNVLPEYRGEDGYRVGRISFNNISAILGTVA VILNCHHQGARSVR SEQ IDShiga toxin REFTIDFSTQQSYVSSLNSIRTEISTPLEHISQGTTSVSVINHTPPGSYF NO: 15subtype 2d AVDIRGLDVYQARFDHLRLIIEQNNLYVAGFVNTATNTFYRFSDFT Subunit AHISVPGVTTVSMTTDSSYTTLQRVAALERSGMQISRHSLVSSYLALM (Stx2dA)EFSGNTMTRDASRAVLRFVTVTAEALRFRQIQREFRQALSETAPVY variant 3TMTPGDVDLTLNWGRISNVLPEYRGEDGYRVGRISFNNISAILGTVA VILNCHHQGARSVR SEQ IDShiga toxin QEFTIDFSTQQSYVSSLNSIRTAISTPLEHISQGATSVSVINHTPPGSYI NO: 16subtype 2e SVGIRGLDVYQERFDHLRLIIERNNLYVAGFVNTTTNTFYRFSDFAH Subunit AISLPGVTTISMTTDSSYTTLQRVAALERSGMQISRHSLVSSYLALMEF (Stx2eA)SGNTMTRDASRAVLRFVTVTAEALRFRQIQREFRLALSETAPVYTM variant 1TPEDVDLTLNWGRISNVLPEYRGEAGVRVGRISFNNISAILGTVAVIL NCHHQGARSVR SEQ IDShiga toxin QEFTIDFSTQQSYVSSLNSIRTAISTPLEHISQGATSVSVINHTPPGSYI NO: 17subtype 2e SVGIRGLDVYQAHFDHLRLIIEQNNLYVAGFVNTATNTFYRFSDFA Subunit AHISLPGVTTISMTTDSSYTTLQRVAALERSGMQISRHSLVSSYLALM (Stx2eA)EFSGNTMTREASRAVLRFVTVTAEALRERQIQREFRQALSETAPVYT variant 2MTPEDVDETLNWGRISNVEPEYRGEDGVRVGRISFNNISAILGTVAV ILNCHHQGARSVR SEQ IDShiga toxin DEFTVDFSSQKSYVDSLNSIRSAISTPLGNISQGGVSVSVINHVPGGN NO: 18subtype 2f YISLNVRGLDPYSERFNHLRLIMERNNLYVAGFINTETNTFYRFSDFS Subunit AHISVPDVITVSMTTDSSYSSLQRIADLERTGMQIGRHSLVGSYLDLM (Stx2fA)EFRGRSMTRASSRAMLRFVTVIAEALRFRQIQRGFRPALSEASPLYTMTAQDVDLTLNWGRISNVLPEYRGEEGVRIGRISFNSLSAILGSVAV ILNCHSTGSYSVR SEQ IDT-cell epitope VTEHDTLLY NO: 19 C1 SEQ ID T-cell epitope GLDRNSGNYNO: 20 C1-2 SEQ ID T-cell epitope NLVPMVATV NO: 21 C2 SEQ IDT-cell epitope GVMTRGRLK NO: 22 C3 SEQ ID T-cell epitope VYALPLKMLNO: 23 C24 SEQ ID T-cell epitope QYDPVAALF NO: 24 C24-2 SEQ IDT-cell epitope GILGFVFTL NO: 25 F2 SEQ ID T-cell epitope ILRGSVAHKNO: 26 F3 SEQ ID T-cell epitope CLGGLLTMV NO: 27 E2 SEQ IDT-cell epitope- NLVPMVATVRRNLVPMVATVRRNLVPMVATV NO: 28 polypeptide (C2)₃SEQ ID Shiga toxin AEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLNO: 29 effector FAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSpolypeptide HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLM SLT-1A-DI-SHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSY FRVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSV ALILNSHHHASAVAA SEQ IDShiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 30effector FAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS polypeptideHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLM SLT-1A-DI-1SHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISGSINAILGS VALILNSHHHASAVAA SEQ IDShiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 31effector FAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS polypeptideHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLM SLT-1A-DI-2SHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISGSINAILGS VALILNSHHHASAVAA SEQ IDShiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 32effector FAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS polypeptideHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLM SLT-1A-DI-3SHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISGSINAILGS VALILNSHHHASAVAA SEQ IDShiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQPISSGGTSLLMIDSGSGDN NO: 33effector LFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAD polypeptideFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLD SLT-1A-DI-4LMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAIL GSVALILNCHHHASAVAA SEQ IDShiga toxin AEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 34effector FAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS polypeptideHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLM SLT-1A-DI-SHSATSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRS FR inactiveYVMTAEDVDLTINWGRLSSVLPDYHGQDSVRVGRISFGSINAILGS VALILNSHHHASAVAA SEQ IDShiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 35effector FAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS polypeptideHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLM SLT-1A-DI-1SHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGAS inactiveYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGS VALILNSHHHASAVAA SEQ IDShiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 36effector FAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS polypeptideHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLM SLT-1A-DI-2SHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGAS inactiveYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGS VALILNSHHHASAVAA SEQ IDShiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO 37effector FAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS polypeptideHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLM SLT-1A-DI-3SHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGAS inactiveYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGS VALILNSHHHASAVAA SEQ IDShiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDN NO: 38effector LFAVDNRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVEYRFAD polypeptideFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLD SLT-1A-DI-4LMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSG inactiveRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAIL GSVALILNCHHHASAVAA SEQ IDlight chain QDISNYLA NO: 39 ABR1 SEQ ID light chain LLIYYTSILHS NO: 40ABR2 SEQ ID light chain QQGNTLPW NO: 41 ABR3 SEQ ID heavy chainYTFTSYWLH NO: 42 ABR1 SEQ ID heavy chain WIGYINPRNDYTEY NO: 43 ABR2SEQ ID heavy chain RRDITTFY NO: 44 ABR3 SEQ ID light chainQSVLYSANHKNYLA NO: 45 ABR1 SEQ ID light chain LLIYWASTRES NO: 46 ABR2SEQ ID light chain HQYLSSW NO: 47 ABR3 SEQ ID heavy chain YEFSRSWMNNO: 48 ABR1 SEQ ID heavy chain WVGRIYPGDGDTNYSGKF NO: 49 ABR2 SEQ IDheavy chain RDGSSWDWYFDV NO: 50 ABR3 SEQ ID light chain QSIVHSVGNTFLENO: 51 ABR1 SEQ ID light chain LLIYKVSNRFS NO: 52 ABR2 SEQ IDlight chain FQGSQFPY NO: 53 ABR3 SEQ ID heavy chain GYRFTNYWIH NO: 54CDR1 SEQ ID heavy chain GINPGNNYATYRRKFQG NO: 55 CDR2 SEQ ID heavy chainEGYGNYGAWFAY NO: 56 CDR3 SEQ ID light chain RSSQSLANSYGNTFLS NO: 57 CDR1SEQ ID light chain GISNRFS NO: 58 CDR2 SEQ ID light chain LQGTHQPYTNO: 59 CDR3 SEQ ID heavy chain GFAFSIYDMS NO: 60 CDR1 SEQ ID heavy chainYISSGGGTTYYPDTVKG NO: 61 CDR2 SEQ ID heavy chain HSGYGTHWGVLFAY NO: 62CDR3 SEQ ID light chain RASQDISNYLA NO: 63 CDR1 SEQ ID light chainYTSILHS NO: 64 CDR2 SEQ ID light chain QQGNTLPWT NO: 65 CDR3 SEQ IDheavy chain GYTFTDYYIT NO: 66 CDR1 SEQ ID heavy chain WIYPGSGNTKYNEKFNO: 67 CDR2 SEQ ID heavy chain YGNYWFAY NO: 68 CDR3 SEQ ID light chainKASQSVDFDGDSYMN NO: 69 CDR1 SEQ ID light chain AASNLES NO: 70 CDR2SEQ ID light chain QQSNEDPWT NO: 71 CDR3 SEQ ID heavy chain YTFTTYWMHNO: 72 CDR1 SEQ ID heavy chain WIGYINPSTGYTDY NO: 73 CDR2 SEQ IDheavy chain TRRGPSYGNHGAWFPY NO: 74 CDR3 SEQ ID light chain ENVDTYVSNO: 75 CDR1 SEQ ID light chain LIAYGASNRYT NO: 76 CDR2 SEQ IDlight chain GQSYRYPP NO: 77 CDR3 SEQ ID heavy chain GYTFTGYYMH NO: 78CDR1 SEQ ID heavy chain WIDPNSGATTYAQKF NO: 79 CDR2 SEQ ID heavy chainKTTQTTWGFPF NO: 80 CDR3 SEQ ID light chain RASQGVYQWLA NO: 81 CDR1SEQ ID light chain KASHLYN NO: 82 CDR2 SEQ ID light chain QQLNSYPLTNO: 83 CDR3 SEQ ID heavy chain GYTFTDYWMH NO: 84 CDR1 SEQ ID heavy chainWIGYINPNTAYTDY NO: 85 CDR2 SEQ ID light chain KASENVDSFVS NO: 86 CDR1SEQ ID light chain GASNRYT NO: 87 CDR2 SEQ ID light chain GQNYRYPLTNO: 88 CDR3 SEQ ID heavy chain FSLISYGVH NO: 89 ABR1 SEQ ID heavy chainWLGVIWRGGSTDY NO: 90 ABR2 SEQ ID heavy chain KTLITTGYAMDY NO: 91 ABR3SEQ ID light chain EDIYNRLA NO: 92 ABR1 SEQ ID light chain LLISGATSLETGNO: 93 ABR2 SEQ ID light chain QQYWSTP NO: 94 ABR3 SEQ ID heavy chainFTFNSFAMS NO: 95 ABR1 SEQ ID heavy chain WVSAISGSGGGTYY NO: 96 ABR2SEQ ID heavy chain KDKILWFGEPVFDY NO: 97 ABR3 SEQ ID light chainQSVSSYLA NO: 98 ABR1 SEQ ID light chain LLIYDASNRAT NO: 99 ABR2 SEQ IDlight chain QQRSNWPP NO: 100 ABR3 SEQ ID heavy chain FSLTSYGVH NO: 101ABR1 SEQ ID heavy chain WIGVMWRGGSTDY NO: 102 ABR2 SEQ ID heavy chainKSMITTGFVMDS NO: 103 ABR3 SEQ ID light chain EDIYNRLT NO: 104 ABR1SEQ ID light chain LLISGATSLET NO: 105 ABR2 SEQ ID light chain QQYWSNPYNO: 106 ABR3 SEQ ID heavy chain FDFSRSWMN NO: 107 ABR1 SEQ IDheavy chain WIGEINPDSSTINY NO: 108 ABR2 SEQ ID heavy chain RYGNWFPYNO: 109 ABR3 SEQ ID light chain QNVDTNVA NO: 110 ABR1 SEQ ID light chainALIYSASYRYS NO: 111 ABR2 SEQ ID light chain QQYDSYPL NO: 112 ABR3 SEQ IDheavy chain GTFSSYAFS NO: 113 ABMR1 SEQ ID heavy chain WMGRVIPFLGIANSNO: 114 ABR2 SEQ ID heavy chain RDDIAALGPFDY NO: 115 ABR3 SEQ IDlight chain QGISSWLA NO: 116 ABR1 SEQ ID light chain SLIYAASSLQS NO: 117ABR2 SEQ ID light chain QQYNSYPR NO: 118 ABR3 SEQ ID heavy chainYTFTDYWMQ NO: 119 ABR1 SEQ ID heavy chain WIGTIYPGDGDTGY NO: 120 ABR2SEQ ID heavy chain RGDYYGSNSLDY NO: 121 ABR3 SEQ ID light chain QDVSTVVANO: 122 ABR1 SEQ ID light chain RLIYSASYRYI NO: 123 ABR2 SEQ IDlight chain QQHYSPPY NO: 124 ABR3 SEQ ID heavy chain GFSLTSYGVH NO: 125CDR1 SEQ ID heavy chain VMWRGGSTDYNAAFMS NO: 126 CDR2 SEQ ID heavy chainSMITTGFVMDS NO: 127 CDR3 SEQ ID light chain KASEDIYNRLT NO: 128 CDR1SEQ ID light chain GATSLET NO: 129 CDR2 SEQ ID light chain QQYWSNPYTNO: 130 CDR3 SEQ ID heavy chain GFSLISYGVH NO: 131 CDR1 SEQ IDheavy chain VIWRGGSTDYNAAFMS NO: 132 CDR2 SEQ ID heavy chain TLITTGYAMDYNO: 133 CDR3 SEQ ID light chain KASEDIYNRLA NO: 134 CDR1 SEQ IDlight chain GATSLET NO: 135 CDR2 SEQ ID light chain QQYWSTPT NO: 136CDR3 SEQ ID heavy chain GFDFSRSWMN NO: 137 CDR1 SEQ ID heavy chainEINPDSSTINYTTSLKD NO: 138 CDR2 SEQ ID heavy chain YGNWFPY NO: 139 CDR3SEQ ID light chain KASQNVDTNVA NO: 140 CDR1 SEQ ID light chain SASYRYSNO: 141 CDR2 SEQ ID light chain QQYDSYPLT NO: 142 CDR3 SEQ IDheavy chain FDFSRYWMS NO: 143 ABR1 SEQ ID heavy chain WIGEINPTSSTINFNO: 144 ABR2 SEQ ID heavy chain RGNYYRYGDAMDY NO: 145 ABR3 SEQ IDlight chain KSVSTSGYSYLH NO: 146 ABR1 SEQ ID light chain LLIYLASNLESNO: 147 ABR2 SEQ ID light chain QHSRELPF NO: 148 ABR3 SEQ ID heavy chainSTFTTYWIH NO: 149 ABR1 SEQ ID heavy chain WIGYINPNTGYTEY NO: 150 ABR2SEQ ID heavy chain VRFITVVGG NO: 151 ABR3 SEQ ID light chain SSVSSSHLHNO: 152 ABR1 SEQ ID light chain LWIYSTSNLAS NO: 153 ABR2 SEQ IDlight chain HQYHRSPL NO: 154 ABR3 SEQ ID heavy chain FSLTTYGIGVG NO: 155ABR1 SEQ ID heavy chain WLTHIWWNDNKYY NO: 156 ABR2 SEQ ID heavy chainYGYTY NO: 157 ABR3 SEQ ID light chain QSLLYSNGNTYLH NO: 158 ABR1 SEQ IDlight chain LLIYKLSNRFS NO: 159 ABR2 SEQ ID light chain SQSTHVPW NO: 160ABR3 SEQ ID heavy chain FNIKDTYIH NO: 161 ABR1 SEQ ID heavy chainWVARIYPTNGYTRY NO: 162 ABR2 SEQ ID heavy chain RWGGDGFYAMDY NO: 163 ABR3SEQ ID light chain QDVNTAVA NO: 164 ABR1 SEQ ID light chain LLIYSASFLYSNO: 165 ABR2 SEQ ID light chain QQHYTTPP NO: 166 ABR3 SEQ ID heavy chainRWGGDGFYAMDV NO: 167 ABR3 SEQ ID heavy chain YSFTSYWIA NO: 168 ABR1SEQ ID heavy chain YMGLIYPGDSDTKY NO: 169 ABR2 SEQ ID heavy chainRHDVGYCSSSNCAKWPEYFQH NO: 170 ABR3 SEQ ID light chain SSNIGNNYVS NO: 171ABR1 SEQ ID light chain LLIYGHTNRPA NO: 172 ABR2 SEQ ID light chainAAWDDSLSGW NO: 173 ABR3 SEQ ID heavy chain YPFTNYGMN NO: 174 ABR1 SEQ IDheavy chain WMGWINTSTGESTF NO: 175 ABR2 SEQ ID heavy chain RWEVYHGYVPYNO: 176 ABR3 SEQ ID light chain QDVYNAVA NO: 177 ABR1 SEQ ID light chainLLIYSASSRYT NO: 178 ABR2 SEQ ID light chain QQHFRTPF NO: 179 ABR3 SEQ IDheavy chain ITFSINTMG NO: 180 ABR1 SEQ ID heavy chain LVALISSIGDTYYANO: 181 ABR2 SEQ ID heavy chain KRFRTAAQGTDY NO: 182 ABR3 SEQ IDheavy chain GFNIKDTYIH NO: 183 CDR1 SEQ ID heavy chain RIYPTNGYTRYADSVKGNO: 184 CDR2 SEQ ID heavy chain WGGDGFYAMDY NO: 185 CDR3 SEQ IDlight chain RASQDVNTAVA NO: 186 CDR1 SEQ ID light chain SASFLYS NO: 187CDR2 SEQ ID light chain QQHYTTPPT NO: 188 CDR3 SEQ ID heavy chainGFNIKDTYIH NO: 189 CDR1 SEQ ID heavy chain RIYPTNGYTRYADSVKG NO: 190CDR2 SEQ ID heavy chain WGGDGFYAMDV NO: 191 CDR3 SEQ ID light chainRASQDVNTAVA NO: 192 CDR1 SEQ ID light chain SASFLYS NO: 193 CDR2 SEQ IDlight chain QQHYTTPPT NO: 194 CDR3 SEQ ID heavy chain GYSFTSYWIA NO: 195CDR1 SEQ ID heavy chain LIYPGDSDTKYSPSFQG NO: 196 CDR2 SEQ IDheavy chain HDVGYCSSSNCAKWPEYFQH NO: 197 CDR3 SEQ ID light chainSGSSSNIGNNYVS NO: 198 CDR1 SEQ ID light chain GHTNRPA NO: 199 CDR2SEQ ID light chain AAWDDSLSGWV NO: 200 CDR3 SEQ ID heavy chainGITFSINTMG NO: 201 CDR1 SEQ ID heavy chain LISSIGDTYYADSVKG NO: 202 CDR2SEQ ID heavy chain FRTAAQGTDY NO: 203 CDR3 SEQ ID heavy chain FTFSDSWIHNO: 204 ABR1 SEQ ID heavy chain WVAWISPYGGSTYY NO: 205 ABR2 SEQ IDheavy chain RRHWPGGFDY NO: 206 ABR3 SEQ ID light chain QDVSTAVA NO: 207ABR1 SEQ ID light chain LLIYSASFLYS NO: 208 ABR2 SEQ ID light chainQQYLYHPA NO: 209 ABR3 SEQ ID heavy chain YTFTSYVMH NO: 210 ABR1 SEQ IDheavy chain WIGYVNPFNDGTKY NO: 211 ABR2 SEQ ID heavy chain RQAWGYPNO: 212 ABR3 SEQ ID light chain ESVEYYGTSLVQ NO: 213 ABR1 SEQ IDlight chain LLIYAASSVDS NO: 214 ABR2 SEQ ID light chain QQSRRVPY NO: 215ABR3 SEQ ID heavy chain YTFTSYDVH NO: 216 ABR1 SEQ ID heavy chainWMGWLHADTGITKF NO: 217 ABR2 SEQ ID heavy chain RERIQLWFDY NO: 218 ABR3SEQ ID light chain QGISSWLA NO: 219 ABR1 SEQ ID light chain SLIYAASSLQSNO: 220 ABR2 SEQ ID light chain QQYNSYPY NO: 221 ABR3 SEQ ID heavy chainDTFSTYAIS NO: 222 ABR1 SEQ ID heavy chain WMGGIIPIFGKAHY NO: 223 ABR2SEQ ID heavy chain RKFHFVSGSPFGMDV NO: 224 ABR3 SEQ ID light chainQSVSSYLA NO: 225 ABR1 SEQ ID light chain LLIYDASNRAT NO: 226 ABR2 SEQ IDlight chain QQRSNWP NO: 227 ABR3 SEQ ID heavy chain FTFSSYIMM NO: 228ABR1 SEQ ID heavy chain WVSSIYPSGGITFY NO: 229 ABR2 SEQ ID heavy chainRIKLGTVTTVDY NO: 230 ABR3 SEQ ID light chain SSDVGGYNYVS NO: 231 ABR1SEQ ID light chain LMIYDVSNRPS NO: 232 ABR2 SEQ ID light chain SSYTSSSTRNO: 233 ABR3 SEQ ID heavy chain GFNIKDYFLH NO: 234 CDR1 SEQ IDheavy chain WINPDNGNTVYDPKFQG NO: 235 CDR2 SEQ ID heavy chainRDYTYEKAALDY NO: 236 CDR3 SEQ ID light chain RASGNIYNYLA NO: 237 CDR1SEQ ID light chain DAKTLAD NO: 238 CDR2 SEQ ID light chain QHFWSLPFTNO: 239 CDR3 SEQ ID heavy chain YTFTSYVMH NO: 240 CDR1 SEQ IDheavy chain YVNPFNDGTKYNEMF NO: 241 CDR2 SEQ ID heavy chain QAWGYPNO: 242 CDR3 SEQ ID light chain RATESVEYYGTSLVQ NO: 243 CDR1 SEQ IDlight chain AASSVDS NO: 244 CDR2 SEQ ID light chain QQSRRVPYT NO: 245CDR3 SEQ ID linker 1 EFPKPSTPPGSSGGAP NO: 246 SEQ ID linker 1 withEFPKPSTPPGSSGGAPGILGFVFTL NO: 247 extension SEQ ID linker 2 GGGGSGGNO: 248 SEQ ID linker 3 GGGGSGGGGSGGGGSGGGGSGGGGS NO: 249 SEQ IDlinker 4 GSTSGSGKPGSGEGS NO: 250 SEQ ID linker 5 GGGGS NO: 251 SEQ IDexemplary MAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 252cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA moleculeDFSHYTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYL SLTA-1A-DI-DLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLS FR::scFv1::C2GRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSNLVPMVATV SEQ ID exemplaryMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGD NO: 253 cell-targetingNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFNNRTNNVFYRFA moleculeDFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYL inactive SLT-DLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLS 1A-DI-GRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAI 4::scFv6::(C2)LGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLV 3QPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKNLVPMVATVRRNINPMVATVRRNLVPMVATV SEQ ID exemplaryMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 254 cell-targetingNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA moleculeDFSHYTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYL inactive SLT-DLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLS 1A-DI-GASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAI 1::scFv8::C2LGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIVLTQSPASLAVSLGQRATISCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPARFSGSGSGTDFSLTIHPVEEDDIAMYFCQQSRRVPYTFGGGTKLEIKGGGGSEVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYVNPFNDGTKYNEMFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARQAWGYPWGQGTLVTVSANLVPMVATV SEQ ID exemplaryMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 255 cell-targetingNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA moleculeDFSHYTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYL SLT-1A-DI-DLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLS 1::scFv8::C2GASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIVLTQSPASLAVSLGQRATISCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPARFSGSGSGTDFSLTIHPVEEDDIAMYFCQQSRRVPYTFGGGTKLEIKGGGGSEVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYVNPFNDGTKYNEMFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARQAWGYPWGQGTLVTVSANLVPMVATV SEQ ID reference cell-MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 256 targetingNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 29DFSHYTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIVLTQSPASLAVSLGQRATISCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPARFSGSGSGTDFSLTIHPVEEDDIAMYFCQQSRRVPYTFGGGTKLEIKGGGGSEVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYVNPFNDGTKYNEMFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARQAWGYPWGQGTLVTVSA SEQ ID reference cell-MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 257 targetingNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 30DFSHYTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIVLTQSPASLAVSLGQRATISCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPARFSGSGSGTDFSLTIHPVEEDDIAMYFCQQSRRVPYTFGGGTKLEIKGGGGSEVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYVNPFNDGTKYNEMFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARQAWGYPWGQGTLVTVSA SEQ ID reference cell-MAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 258 targetingNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA moleculeDFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYL SLTA-1A-DI-DLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLS FR::scFv1GRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPLKKISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSA SEQ ID cell-targetingMVTEHDTLLYKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL NO: 259 molecule 1LMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNR C1::SLT-TNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQIN 1A::scFv1RHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID cell-targetingMGLDRNSGNYKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTS NO: 260 molecule 2LLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVN C1-2::SLT-RTNNVFYRFADFSHVTFPGTTAVFLSGDSSYTTLQRVAGISRTGMQI 1A::scFv1NRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID cell-targetingMGVMTRGRLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL NO: 261 molecule 3LMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNR C3::SLT-TNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQIN 1A::scFv1RHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID cell-targetingMVYALPLKMLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTS NO: 262 molecule 4LLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVN C24::SLT-RTNNVFYRFADFSHVTFPGTTAVFLSGDSSYTTLQRVAGISRTGMQI 1A::scFv1NRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGD NO: 263 molecule 5NLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA SLT-DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYL 1A::scFv1::C1DLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSVTEHDTLLY SEQ ID cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGD NO: 264 molecule 6NLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA SLT-DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYL 1A::scFv1::C2DLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLS 4-2GRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSQYDPVAALF SEQ ID cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGD NO: 265 molecule 7NLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA SLT-DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYL 1A::scFv1::E2DLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSCLGGLLTMV SEQ ID cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGD NO: 266 molecule 8NLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA SLT-DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYL 1A::scFv1:F3DLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSILRGSVAHK SEQ ID cell-targetingMNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTS NO: 267 molecule 9LLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVN C2::SLT-RTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQI 1A::scFv2NRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGNMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSW GQGSLVTVSS SEQ IDcell-targeting MNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTS NO: 268molecule 10 LLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVN inactiveRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQI C2::SLT-NRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQR 1A::scFv2GFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGNMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSW GQGSLVTVSS SEQ IDcell-targeting MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGD NO: 269molecule 11 NLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA SLT-DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYL 1A::scFv2::C2DLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVS SNLVPMVATV SEQ IDcell-targeting MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGD NO: 270molecule 12 NLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA inactive SLT-DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYL 1A::scFv2::C2DLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVS SNLVPMVATV SEQ IDcell-targeting MGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 271molecule 13 MIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRT F2::SLT-NNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINR 1A::scFv2HSLTTSYLDIMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYYMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDLYNRLTWYQQKPGKAPKLLISGATSLETGVPSRESGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWG QGSLVTVSS SEQ IDcell-targeting MDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAP NO: 272molecule 14 KLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYscFv3::F2::SL TTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPG T-1AGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNS+RAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSSEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNC HHHASRVAR SEQ IDcell-targeting MQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQ NO: 273molecule 15 GLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSE scFv4::F2::SLDSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGS T-1AGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN CHHHASRVAR SEQ IDcell-targeting MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGD NO: 274molecule 16 NLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA SLT-DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYL 1A::scFv5::C2DLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSNLVPMVAT V SEQ ID cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGD NO: 275 molecule 17NLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA SLT-DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYL 1A::scFv6::F2DLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATF GQGTKVEIKGILGFVFTLSEQ ID cell-targeting MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNO: 276 molecule 18 NLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAinactive SLT- DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYL1A::scFv6::F2 DLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATF GQGTKVEIKGILGFVFTLSEQ ID cell-targeting MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNO: 277 molecule 19 NLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA SLT-DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYL 1A::scFv7::C2DLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDVHWVRQAPGQRLEWMGWLHADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARERIQLWFDYWGQGTLVTVSSNLVPMVATV SEQ ID cell-targetingMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGD NO: 278 molecule 20NLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA SLT-DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYL 1A::scFv1::C2DLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSNLVPMVATV SEQ ID wild-typeKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDN NO: 279 Shiga toxinLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAD effectorFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLD polypeptideLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSG (SLT-1A-WT)RSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAIL GSVALILNCHHHASRVAR SEQ IDreference cell- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGD NO: 280targeting NLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 21DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID reference cell-MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGD NO: 281 targetingNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 22DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVS S SEQ ID reference cell-MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGD NO: 282 targetingNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 23DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVS S SEQ ID reference cell-MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGD NO: 283 targetingNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 24DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS SEQ ID reference cell-MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGD NO: 284 targetingNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 25DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSCAASGFTESDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATF GQGTKVEIK SEQ IDreference cell- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGD NO: 285targeting NLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 26DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSCAASGFTESDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATF GQGTKVEIK SEQ IDreference cell- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGD NO: 286targeting NLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 27DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDVHWVRQAPGQRLEWMGWLHADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARERIQLWFDYWGQGTLVTVSS SEQ ID reference cell-MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGD NO: 287 targetingNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 28DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLMQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQGLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEDSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISN PPTFGAGTKLELK SEQ IDexemplary VTEHDTLLYAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 288cell-targeting MIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNmolecule 1 NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVYVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDISTYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSHFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID exemplaryGLDRNSGNYAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 289 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 2NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDISTYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSHFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID exemplaryGVMTRGRLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 290 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 3NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDISTYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSHFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID exemplaryVYALPLKMLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 291 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 4NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDISTYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSHFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID exemplaryNLVPMVATVAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 292 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 5NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDISTYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQ GSLVTVSS SEQ ID exemplaryGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLM NO: 293 cell-targetingIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNN molecule 6VFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSASTSLTQSARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDISTYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQ GSLVTVSS SEQ ID exemplaryDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKL NO: 294 cell-targetingLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLOPEDFATYYCQQHYTT molecule 7PPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGEYAMDVWGQGTLVTVSSEFPKPSTPPGSSGGAPGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHH HASAVAA SEQ ID exemplaryQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQG NO: 295 cell-targetingLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEDS molecule 8AVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSH HHASAVAA SEQ IDexemplary AEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 296cell-targeting FAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSmolecule 9 HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSVTEHDTLLY SEQ ID exemplaryAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 297 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 10HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSVTEHDTLLY SEQ ID exemplaryAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 298 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 11HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSQYDPVAALF SEQ ID exemplaryAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 299 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 12HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSCLGGLLTMV SEQ ID exemplaryAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 300 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 13HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSILRGSVAHK SEQ ID exemplaryAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 301 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 14HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSNL VPMVATV SEQ ID exemplaryAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 302 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 15HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSNLVPMVATV SEQ ID exemplaryAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 303 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 16HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQG TKVEIKGILGFVFTL SEQ IDexemplary AEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 304cell-targeting FAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSmolecule 17 HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDVHWVRQAPGQRLEWMGWLHADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARERIQLWFDYWGQGTLVTVSSNLVPMVATV SEQ ID exemplaryAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 305 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 18HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSNLVPMVATV SEQ ID exemplaryAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 306 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 19HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQGLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEDSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELK NLVPMVATV SEQ IDexemplary AEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 307cell-targeting FAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSmolecule 20 HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARAQLRPNYWYFDVWGAGTTVTVSSGGGGSDIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKNLVPMVATV SEQ ID exemplaryAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 308 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 21HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAGSTSGSGKPGSGEGSTKGQIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEI KNLVPMVATV SEQ IDexemplary AEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 309cell-targeting FAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSmolecule 22 HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTDY WGQGTQVTVSSAHHSEDNLVPMVATVSEQ ID exemplary AEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLNO: 310 cell targeting FAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSmolecule 23 HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIELTQSPSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPTFGGGTKLEIKGSTSGSGKPGSGEGSKVQLQESGPSLVQRSQRLSITCTVSGFSLISYGVHWVRQSPGKGLEWLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTAIYFCAKTLITTGYAMDYWGQGTTVTVSSNLVPMVAT V SEQ ID exemplaryAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 311 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 24HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAAHHSEDPSSKAPKAPEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTDY WGQGTQVTVSSNLVPMVATVSEQ ID exemplary AEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLNO: 312 cell-targeting FAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSmolecule 25 HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPASVSDVPRDLEVVAATPTSLLISWCRQRCADSYRITYGETGGNSPVQEFTVPGSWKTATISGLKPGVDYTITVYVVTHYYGWDRYSHPISINYRTGSNLVPMVATV SEQ ID exemplaryASVSDVPRDLEVVAATPTSLLISWCRQRCADSYRITYGETGGNSPV NO: 313 cell-targetingQEFTVPGSWKTATISGLKPGVDYTITVYVVTHYYGWDRYSHPISIN molecule 26YRTGSEFPKPSTPPGSSGGAPAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAANLVPMVA TV SEQ ID exemplaryAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 314 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 27HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSHSTLTNINPMVATV SEQ ID exemplaryNLVPMVATVAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 315 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 28NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAGGGGSGGDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID exemplaryNLVPMVATVAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 316 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 29NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRESGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQ GSLVTVSS SEQ ID exemplaryNLVPMVATVAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 317 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 30NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS SEQ ID exemplaryGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLM NO: 318 cell-targetingIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNN molecule 31VFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNFAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQY LYHPATFGQGTKVEIK SEQ IDexemplary NLVPMVATVAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 319cell-targeting MIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNmolecule 32 NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAGGGGSGGDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDVHWVRQAPGQRLEWMGWLHADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARERIQLWFDYWGQGTLVTVSS SEQ ID exemplaryNLVPMVATVAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 320 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 33NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID exemplaryNLVPMVATVAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 321 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 34NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQGLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEDSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFG AGTKLELK SEQ IDexemplary NLVPMVATVAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 322cell-targeting MIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNmolecule 35 NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAGGGGSGGQVQLVQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARAQLRPNYWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNP PTFGAGTKLELK SEQ IDexemplary APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYM NO: 323cell-targeting PKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLmolecule 36 ELKGSETTFMCEYADETATIVEFLNRWITFCQSHSTLTEFPKPSTPPGSSGGAPNLVPMVATVAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLIMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVFVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID exemplaryGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLM NO: 324 cell-targetingIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNN molecule 37VFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSHSTLT SEQ ID exemplaryMNLVPMVATVAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTS NO: 325 cell-targetingLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNR molecule 38TNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWG QGSLVTVSS SEQ ID exemplaryMNLVPMVATVAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTS NO: 326 cell-targetingLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNR molecule 39TNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWG QGSLVTVSS SEQ ID exemplaryMAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 327 cell-targetingNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNYFYRFA molecule 40DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVS SNLVPMVATV SEQ IDexemplary MAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 328cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 41 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMIQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVS SNLVPMVATV SEQ IDexemplary MGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 329cell targeting MIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVFGFVNRTNmolecule 42 NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQ GSLVTVSS SEQ ID exemplary-MDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAP NO: 330 cell-targetingKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHY molecule 43TTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARTYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSSEFPKPSTPPGSSGGAPGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLOTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSH HHASAVAA SEQ IDexemplary MQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQ NO: 331cell-targeting GLEWIGATYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEmolecule 44 DSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYREADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN SHHHASAVAA SEQ IDexemplary MAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 332cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 45 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMIQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSNLVPMVATV SEQ ID exemplaryMAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 333 cell-targetingNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 46DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATF GQGTKVEIKGILGFVFTLSEQ ID exemplary MAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNO: 334 cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 47 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATF GQGTKVEIKGILGFVFTLSEQ ID exemplary MAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNO: 335 cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 48 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDVHWVRQAPGQRLEWMGWLHADTGITKESQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARERIQLWFDYWGQGTLVTVSSNLVPMVATV SEQ ID exemplaryMAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 336 cell-targetingNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 49DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPLKKISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSNLVPMVATV SEQ ID exemplaryQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQG NO: 337 cell-targetingLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEDS molecule 50AVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSH HHASAVAA SEQ IDexemplary MQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQ NO: 338cell-targeting GLEWIGAIYPGGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSE molecule 51DSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN SHHHASAVAA SEQ IDexemplary MDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAP NO: 339cell-targeting KLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYmolecule 52 TTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSSEFPKPSTPPGSSGGAPGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSH HHASAVAAKDEL SEQ IDexemplary MDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAP NO: 340cell-targeting KLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYmolecule 53 TTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSSEFPKPSTPPGSSGGAPGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSH HHASAVAAKDEL SEQ IDexemplary MDIELTQSPSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGNAPRL NO: 341cell-targeting LISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSmolecule 54 TPTFGGGTKLEIKGSTSGSGKPGSGEGSKVQLQESGPSLVQPSQRLSITCTVSGFSLISYGNHWVRQSPGKGLEWLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTAIYFCAKTLITTGYAMDYWGQGTTVTVSSEFPKPSTPPGSSGGAPGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAV AAKDEL SEQ ID exemplaryMDIELTQSPSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGNAPRL NO: 342 cell-targetingLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWS molecule 55TPTFGGGTKLEIKGSTSGSGKPGSGEGSKVQLQESGPSLVQPSQRLSITCTVSGFSLISYGNHWVRQSPGKGLEWLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTAIYFCAKTLITTGYAMDYWGQGTTVTVSSEFPKPSTPPGSSGGAPGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAV AAKDEL SEQ ID exemplaryMDIVMTQAAPSIPVTPGESVSISCRSSKSLLNSNGNTYLYWFLQRPG NO: 343 cell targetingQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYC molecule 56MQHLEYPFTFGAGTKLELKGSTSGSGKPGSGEGSEVQLQQSGPELIKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGTYYYGSRVFDYWGQGTTLTVSSAEFPKPSTPPGSSGGAPGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSV ALILNSHHHASAVAAKDELSEQ ID exemplary MDIVMTQAAPSIPVTPGESVSISCRSSKSLLNSNGNTYLYWFLQRPG NO: 344cell-targeting QSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCmolecule 57 MQHLEYPFTFGAGTKLELKGSTSGSGKPGSGEGSEVQLQQSGPELIKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGTYYYGSRVFDYWGQGTTLTVSSAEFPKPSTPPGSSGGAPGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSV ALILNSHHHASAVAAKDELSEQ ID exemplary MDIQLTQSPLSLPVTLGQPASISCRSSQSLVHRNGNTYLHWFQQRPG NO: 345cell-targeting QSPRLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSmolecule 58 QSSHVPPTFGAGTRLEIKGSTSGSGKPGSGEGSTKGQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGVNWIKQAPGQGLQWMGWINPNTGEPTFDDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCSRSRGKNEAWFAYWGQGTLVTVSSEFPKPSTPPGSSGGAPGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRFNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALI LNSHHHASAVAAKDEL SEQ IDexemplary MDIQLTQSPLSLPVTLGQPASISCRSSQSLVHRNGNTYLHWFQQRPG NO: 346cell-targeting QSPRLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSmolecule 59 QSSHVPPTFGAGTRLEIKGSTSGSGKPGSGEGSTKGQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGVNWIKQAPGQGLQWMGWINPNTGEPTFDDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCSRSRGKNEAWFAYWGQGTLVTVSSEFPKPSTPPGSSGGAPGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRFNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALI LNSHHHASAVAAKDEL SEQ IDexemplary MEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQ NO: 347cell-targeting RELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAmolecule 60 VYYCKRFRTAAQGTDYWGQGTQVTVSSAHHSEDPSSKAPKAPGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQTQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAKDEL SEQ ID exemplaryMEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQ NO: 348 cell-targetingRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA molecule 61VYYCKRFRTAAQGTDYWGQGTQVTVSSAHHSEDPSSKAPKAPGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQTQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAKDEL SEQ ID exemplaryMEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQ NO: 349 cell-targetingRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA molecule 62VYYCKRFRTAAQGTDYWGQGTQVTVSSAHHSEDPSSKAPKAPGILGFVFTLGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAKDEL SEQ ID exemplaryMEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQ NO: 350 cell-targetingRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA molecule 63VYYCKRFRTAAQGTDYWGQGTQVTVSSAHHSEDPSSKAPKAPGILGFVFTLGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAKDEL SEQ ID exemplaryMAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFY NO: 351 cell-targetingMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVI molecule 64VLELKGSETTFMCEYADETATIVEFLNRWITCQSHSLILTEFPKPSTPPGSSGGAPGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAKDEL SEQ ID exemplaryMAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFY NO: 352 cell-targetingMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVI molecule 65VLELKGSETTFMCEYADETATIVEFLNRWITCQSHSLILTEFPKPSTPPGSSGGAPGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAKDEL SEQ ID exemplaryMQVQLVQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQ NO: 353 cell-targetingGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSE molecule 66DSAVYYCARAQLRPNYWYFDVWGAGTTVTVSSGSTSGSGKPGSGEGSDIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSNAILGSVALILNSH HHASAVAA SEQ ID exemplaryMQVQLVQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQ NO: 354 cell-targetingGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSE molecule 67DSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKSTPPGSSGGAPGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN SHHHASAVAA SEQ IDexemplary MQVQLVQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQ NO: 355cell-targeting GLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEmolecule 68 DSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKSTPPGSSGGAPGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN SHHHASAVAA SEQ IDexemplary MQVQLVQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQ NO: 356cell-targeting GLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEmolecule 69 DSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKSTPPGSSGGAPGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN SHHHASAVAA SEQ IDexemplary MQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGR NO: 357cell-targeting GLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEmolecule 70 DSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAGSTSGSGKPGSGEGSTKGQIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKEFPKPSTPPGSSGGAPGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN SHHHASAVAA SEQ IDexemplary MEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGK NO: 358cell-targeting GLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEmolecule 71 DTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSGSTSGSGKPGSGEGSEIVLTQSPAILSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKEFPKPSTPPGSSGGAPGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSH HHASAVAA SEQ ID exemplaryMEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRL NO: 359 cell-targetingLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNW molecule 72PITFGQGTRLEIKGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSEFPKPSTPPGSSGGAPGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLIMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAI LGSVALILNSHHHASAVAASEQ ID exemplary MQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKP NO: 360cell-targeting WIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFmolecule 73 NPPTFGAGTKLELKSGGGGSGGGGSGGGGSGGGGSGGGGSQAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSEFPKPSTSTPPGSSGGILGFVFTLGAPAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSYLPDYHGQDSVRVGRISFGSI NAILGSVALILNSHHHASAVAASEQ ID exemplary MQAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQ NO: 361cell-targeting GLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEmolecule 74 DSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSGSTSGSGKPGSGEGSQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKSEFPKPSTPPGSSGGAPGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSH HHASAVAA SEQ IDexemplary ASVSDVPRDLEVVAATPTSLLISWCRQRCADSYRITYGETGGNSPV NO: 362cell-targeting QEFTVPGSWKTATISGLKPGVDYTITVYVVTHYYGWDRYSHPISINmolecule 75 YRTGSEFPKPSTPPGSSGGAPGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLYTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID exemplaryMQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQ NO: 363 cell-targetingGLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSE molecule 76DSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAGTGDALVALPEGESVRIADIVPGARPNSDNAIDLKVLDRHGNPVLADRLFHSGEHPVYTVRTVEGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAVIQRSAFSVDCAGFARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYYAKVASVTDAGVQPVYSLRVDTADHAFITNGFVSHATGLTGLNSGLTTNPGVSAWQVNTAYTAGQLVVYNGKTYKCLQPHTSLAGWEPSNVPALWQLQ SEQ ID exemplaryMQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQ NO: 364 cell-targetingGLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSE molecule 77DSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAGTGDALVALPEGESVRIADIVPGARPNSDNAIDLKVLDRHGNPVLADRLFHSGEHPVYTVRTVEGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAVIQRSAFSVDCAGFARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYYAKVASVTDAGVQPVYSLRVDTADHAFITNGFVSHATGLTGLNSGLTTNPGVSAWQVNTAYTAGQLVVYNGKTYKCLQPHTSLAGWEPSNVPALWQLQ SEQ ID exemplaryMQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQ NO: 365 cell-targetingGLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSE molecule 78DSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN SHHHASAVAALDEL SEQ IDexemplary MAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 366cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 79 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVFVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLMQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQGLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSTSEDSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISN PPTFGAGTKLELK SEQ IDexemplary MAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 367cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 80 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVFVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIELTQSPSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPTFGGGTKLEIKGSTSGSGKPGSGEGSKVQLQESGPSLVQPSQRLSITCTVSGFSLISYGVHWVRQSPGKGLEWLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKATNSLQADDTAIYFCAKTLITTGYAMDYWGQGTTVT VSS SEQ ID exemplaryMAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 368 cell-targetingNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 81DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVFVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIELTQSPSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPTFGGGTKLEIKGSTSGSGKPGSGEGSKVQLQESGPSLVQPSQRLSITCTVSGFSLISYGVHWVRQSPGKGLEWLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKATNSLQADDTAIYFCAKTLITTGYAMDYWGQGTTVT VSS SEQ ID exemplaryMAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 369 cell-targetingNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 82DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVFVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPFTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDV WGQGTLVTVSS SEQ IDexemplary MAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 370cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 83 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVFVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPFTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDV WGQGTLVTVSS SEQ IDexemplary MAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 371cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 84 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVFVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIVMTQAAPSIPVTPGESVSISCRSSKSLLNSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGAGTKLELKGSTSGSGKPGSGEGSEVQLQQSGPELIKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGTYYYGSRVF DYWGQGTTLTVSS SEQ IDexemplary MAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 372cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 85 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVFVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIVMTQAAPSIPVTPGESVSISCRSSKSLLNSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGAGTKLELKGSTSGSGKPGSGEGSEVQLQQSGPELIKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGTYYYGSRVF DYWGQGTTLTVSS SEQ IDexemplary MAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 373cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 86 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVFVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIQLTQSPLSLPVTLGQPASISCRSSQSLVHRNGNTYLHWFQQRPGQSPRLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSSHVPPTFGAGTRLEIKGSTSGSGKPGSGEGSTKGQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGVNWIKQAPGQGLQWMGWINPNTGEPTFDDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCSRSRGKNEAW FAYWGQGTLVTVSS SEQ IDexemplary MAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 374cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 87 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVFVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIQLTQSPLSLPVTLGQPASISCRSSQSLVHRNGNTYLHWFQQRPGQSPRLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSSHVPPTFGAGTRLEIKGSTSGSGKPGSGEGSTKGQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGVNWIKQAPGQGLQWMGWINPNTGEPTFDDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCSRSRGKNEAW FAYWGQGTLVTVSS SEQ IDexemplary MAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 375cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 88 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVFVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAAHHSEDPSSKAPKAPGILGFVFTLEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCK RFRTAAQGTDYWGQGTQVTVSSSEQ ID exemplary MAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNO: 376 cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 89 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVFVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAAHHSEDPSSKAPKAPGILGFVFTLEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCK RFRTAAQGTDYWGQGTQVTVSSSEQ ID exemplary MAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNO: 377 cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 90 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVFVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAAHHSEDPSSKAPKAPGILGFVFTLEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCK RFRTAAQGTDYWGQGTQVTVSSSEQ ID exemplary MAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNO: 378 cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 91 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVFVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAAHHSEDPSSKAPKAPGILGFVFTLEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCK RFRTAAQGTDYWGQGTQVTVSSSEQ ID exemplary MAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNO: 379 cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 92 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVFVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLASVSDVPRDLEVVAATPTSLLISWCRQRCADSYRITYGETGGNSPVQEFTVPGSWKTATISGLKPGVDYTITVYVVTHYYGWDRYSHPISINYRTGS SEQ ID exemplaryMAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 380 cell-targetingNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 93DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVFVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLASVSDVPRDLEVVAATPTSLLISWCRQRCADSYRITYGETGGNSPVQEFTVPGSWKTATISGLKPGVDYTITVYVVTHYYGWDRYSHPISINYRTGS SEQ ID exemplaryVTEHDTLLYKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 381 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 94NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLREVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID exemplaryGLDRNSGNYKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 382 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 95NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLREVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID exemplaryGVMTRGRLKEFFLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 383 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 96NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLREVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID exemplaryVYALPLKMLKEFTLDFSTAKTYVDSLNVIRSAIGFPLQTISSGGTSLL NO: 384 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 97NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLREVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID exemplaryNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 385 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 98NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLREVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQ GSLVTVSS SEQ ID exemplaryGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLM NO: 386 cell-targetingIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNN molecule 99VFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLYWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQ GSLVTVS SEQ ID exemplaryDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKL NO: 387 cell-targetingLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTT molecule 100PPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSSEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLEAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHH HASAVAA SEQ ID exemplaryQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQG NO: 388 cell-targetingLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEDS molecule 101AVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSH HHASAVAA SEQ IDexemplary KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 389cell-targeting FAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSmolecule 102 HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSVTEHDTLLY SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 390 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 103HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSNLVPMVATV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 391 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 104HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSQYDPVAALF SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 392 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 105HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSCLGGLLTMV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 393 cell targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 106HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSILRGSVAHK SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 394 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 107HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSN LVPMVATV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 395 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 108HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARTYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSNLVPMVATV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 396 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 109HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQ GTKVEIKGILGFVFTL SEQ IDexemplary KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 397cell-targeting FAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSmolecule 110 HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITGRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFILTISSLQPEDFATYYCQQYNSYRYTFGQGTKLEIKGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDVHWVRQAPGQRLEWMGWLHADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARERIQLWFDYWCOGTLVTVSSNLVPMVATV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 398 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 111HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITGRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFILTISSLQPEDFATYYCQQYNSYRYTFGQGTKLEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSNLVPMVATV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 399 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 112HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQGLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEDSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELK NLVPMVATV SEQ IDexemplary KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 400cell-targeting FAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSmolecule 113 HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARAQLRPNYWYFDVWGAGTTVTVSSGGGGSDIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKNLVPMVATV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 401 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 114HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAGSTSGSGKPGSGEGSTKGQIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEI KNLVPMVATV SEQ IDexemplary KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 402cell-targeting FAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSmolecule 115 HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTDYWGQGTQVTVSSAHHSEDNLVPMVATV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 403 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 116HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIELTQSPSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPTFGGGTKLEIKGSTSGSGKPGSGEGSKVQLQESGPSLVQPSQRLSITCTVSGFSLISYGVHWVRQSPGKGLEWLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTAIYFCAKTLITTGYAMDYWGQGTTVTVSSNLVPMVA TV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 404 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 117HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAAHHSEDPSSKAPKAPEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTD YWGQGTQVTVSSNLVPMVATVSEQ ID exemplary KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLNO: 405 cell-targeting FAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSmolecule 118 HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPASVSDVPRDLEVVAATPTSLLISWCRQRCADSYRITYGETGGNSPVQEFTVPGSWKTATISGLKPGVDYTITVYVVTHYYGWDRYSHPISINYRTGSNLVPMVATV SEQ ID exemplaryASVSDVPRDLEVVAATPTSLLISWCRQRCADSYRITYGETGGNSPV NO: 406 cell-targetingQEFTVPGSWKTATISGLKPGVDYTITVYVVTHYYGWDRYSHPISIN molecule 119YRTGSEFPKPSTPPGSSGGAPKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAANLVPMVA TV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 407 cell targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 120HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIVLTQSPASLAVSLGQRATISCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPARFSGSGSGTDFSLTIHPVEEDDIAMYFCQQSRRVPYTFGGGTKLEIKGGGGSEVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYVNPFNDGTKYNEMFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARQAWGYPWGQGTLVTVSANLVPMVATV SEQ ID exemplaryNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAGTPLQTISSGGTSLL NO: 408 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 121NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAGGGGSGGDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID exemplaryNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAGTPLQTISSGGTSLL NO: 409 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 122NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEITKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQ GSLVTVSS SEQ ID exemplaryNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAGTPLQTISSGGTSLL NO: 410 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 123NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEITKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS SEQ ID exemplaryGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGISLLM NO: 411 cell-targetingIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNN molecule 124VFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQY LYHPATFGQGTKVEIK SEQ IDexemplary NLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAGTPLQTISSGGTSLL NO: 412cell-targeting MIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNmolecule 125 NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHASAVAAGGGGSGGDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDVHWVRQAPGQRLEWMGWLHADTGITKESQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARERIQLWEDYWGQGTLVTVSS SEQ ID exemplaryNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAGTPLQTISSGGTSLL NO: 413 cell targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 126NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDINTYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID exemplaryNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAGTPLQTISSGGTSLL NO: 414 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 127NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQGLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEDSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVRARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFG AGTKLELK SEQ IDexemplary NLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAGTPLQTISSGGTSLL NO: 415cell-targeting MIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNmolecule 128 NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAGGGGSGGQVQLVQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARAQLRPNYWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLASGWARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNP PTFGAGTKLELK SEQ IDexemplary APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYM NO: 416cell-targeting PKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLmolecule 129 ELKGSETTFMCEYADETATIVEFLNRWITFCQSHSTLTEFPKPSTPPGSSGGAPNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID exemplaryGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLM NO: 417 cell-targetingIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNN molecule 130VFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKITRMLTFKFYMPKKATELKHLQCLEEELKPLLEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSHSTLT SEQ ID exemplaryMNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTS NO: 418 cell-targetingLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNR molecule 131TNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGSGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWG QGSLVTVSS SEQ ID exemplaryMNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTS NO: 419 cell-targetingLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNR molecule 132TNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGSGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWG QGSLVTVSS SEQ ID exemplaryMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 420 cell-targetingNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 133DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEIWDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVS SNLVPMVATV SEQ IDexemplary MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 421cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 134 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEIWDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVS SNLVPMVATV SEQ IDexemplary MGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 422cell-targeting MIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNmolecule 135 NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQ GSLVTVSS SEQ ID exemplary-MDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAP NO: 423 cell-targetingKLLIYSASFLYSGVPSRFSGSRSGTDFILTISSLQPEDFATYYCQQHY molecule 136TTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMINSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSSEFPKTSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTISYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSH HHASAVAA SEQ IDexemplary MQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQ NO: 424cell-targeting GLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEmolecule 137 DSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN SHHHASAVAA SEQ IDexemplary MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 425cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 138 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEIWDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVWRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSNLVPMVATV SEQ ID exemplaryMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 426 cell-targetingNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 139DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEIWDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLOPEDFATYYCQQYLYHPATF GQGTKVEIKGILGFVFTLSEQ ID exemplary MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNO: 427 cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 140 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEIWDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLOPEDFATYYCQQYLYHPATF GQGTKVEIKGILGFVFTLSEQ ID exemplary MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNO: 428 cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 141 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEIWDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDVHWVRQAPGQRLEWMGWLHADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARERIQLWFDYWGQGTLVTVSSNLVPMVATV SEQ ID exemplaryMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 429 cell-targetingNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 142DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEIWDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFNISRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSNIATMVATV SEQ ID exemplaryQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQG NO: 430 cell-targetingLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEDS molecule 143AVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSH HHASAVAA SEQ IDexemplary MQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQ NO: 431cell-targeting GLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEmolecule 144 DSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYIIGQDSVRVGRISFGSINAILGSNALILN SHHHASAVAA SEQ IDexemplary MDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAP NO: 432cell-targeting KLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYmolecule 145 TTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSSEFPKPSTPPGSSGGAPGILGFVTTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSH HHASAVAAKDEL SEQ IDexemplary MDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAP NO: 433cell-targeting KLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYmolecule 146 TTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSSEFPKPSTPPGSSGGAPGILGFVTTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSH HHASAVAAKDEL SEQ IDexemplary MDIELTQSPSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGNAPRL NO: 434cell-targeting LISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSmolecule 147 TPTFGGGTKLEIKGSTSGSGKPGSGEGSKVQLQESGPSLVQPSQRLSITCTVSGFSLISYGVHWVRQSPGKGLEWLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTAIYFCAKTLITTGYAMDYWGQGTTVTVSSEFPKPSTPPGSSGGAPGILGFVFILKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSGHHASAV AAKDEL SEQ ID exemplaryMDIELTQSPSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGNAPRL NO: 435 cell-targetingLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWS molecule 148TPTFGGGTKLEIKGSTSGSGKPGSGEGSKVQLQESGPSLVQPSQRLSITCTVSGFSLISYGVHWVRQSPGKGLEWLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTAIYFCAKTLITTGYAMDYWGQGTTVTVSSEFPKPSTPPGSSGGAPGILGFVFILKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSGHHASAV AAKDEL SEQ ID exemplaryMDIVMTQAAPSIPVTPGESVSISCRSSKSLLNSNGNTYLYWFLQRPG NO: 436 cell-targetingQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYC molecule 149MQHLEYPFTFGAGTKLELKGSTSGSGKPGSGEGSEVQLQQSGPELIKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGTYYYGSRVFDYWGQGTTLTVSSAEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSV ALILNSHHHASAVAAKDELSEQ ID exemplary MDIVMTQAAPSIPVTPGESVSISCRSSKSLLNSNGNTYLYWFLQRPG NO: 437cell-targeting QSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCmolecule 150 MQHLEYPFTFGAGTKLELKGSTSGSGKPGSGEGSEVQLQQSGPELIKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGTYYYGSRVFDYWGQGTTLTVSSAEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSV ALILNSHHHASAVAAKDELSEQ ID exemplary MDIQLTQSPLSLPVTLGQPASISCRSSQSLVHRNGNTYLHWFQQRPG NO: 438cell-targeting QSPRLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSmolecule 151 QSSHVPPTFGAGTRIEIKGSTSGSGKPGSGEGSTKGQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGVNWIKQAPGQGLQWMGWINPNTGEPTFDDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCSRSRGKNEAWFAYWGQGTLVTVSSEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALI LNSHHHASAVAAKDEL SEQ IDexemplary MDIQLTQSPLSLPVTLGQPASISCRSSQSLVHRNGNTYLHWFQQRPG NO: 439cell-targeting QSPRLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSmolecule 152 QSSHVPPTFGAGTRIEIKGSTSGSGKPGSGEGSTKGQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGVNWIKQAPGQGLQWMGWINPNTGEPTFDDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCSRSRGKNEAWFAYWGQGTLVTVSSEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALI LNSHHHASAVAAKDEL SEQ IDexemplary MEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQ NO: 440cell-targeting RELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAmolecule 153 VYYCKRFRTAAQGTDYWGQGTQVTVSSAHHSEDPSSKAPKAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSRVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAKDEL SEQ ID exemplaryMEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQ NO: 441 cell-targetingRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA molecule 154VYYCKRFRTAAQGTDYWGQGTQVTVSSAHHSEDPSSKAPKAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSRVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAKDEL SEQ ID exemplaryMEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQ NO: 442 cell-targetingRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA molecule 155VYYCKRFRTAAQGTDYWGQGTQVTVSSAHHSEDPSSKAPKAPGILGFVFTLGILGFVFTLKEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAKDEL SEQ ID exemplaryMEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQ NO: 443 cell-targetingRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA molecule 156VYYCKRFRTAAQGTDYWGQGTQVTVSSAHHSEDPSSKAPKAPGILGFVFTLGILGFVFTLKEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAKDEL SEQ ID exemplaryMAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFY NO: 444 cell-targetingMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVI molecule 157VLELKGSETTFMCEYADETATIVEFLNRWITFCQSHSTLTEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAKDEL SEQ ID exemplaryMAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFY NO: 445 cell-targetingMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVI molecule 158VLELKGSETTFMCEYADETATIVEFLNRWITFCQSHSTLTEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAKDEL SEQ ID exemplaryMQVQLVQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQ NO: 446 cell-targetingGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMWLSSLTSE molecule 159DSAVYYCARAQLRPNYWYFDVWGAGTTVTVSSGSTSGSGKPGSGEGSDIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSH HHASAVAA SEQ IDexemplary MQVQLVQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQ NO: 447cell-targeting GLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMWLSSLTSEmolecule 160 DSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN SHHHASAVAA SEQ IDexemplary MQVQLVQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQ NO: 448cell-targeting GLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMWLSSLTSEmolecule 161 DSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN SHHHASAVAA SEQ IDexemplary MQVQLVQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQ NO: 449cell targeting GLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMWLSSLTSEmolecule 162 DSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN SHHHASAVAA SEQ IDexemplary MQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGR NO: 450cell-targeting GLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEmolecule 163 DSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAGSTSGSGKPGSGEGSTKGQIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN SHHHASAVAA SEQ IDexemplary MEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGK NO: 451cell-targeting GLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEmolecule 164 DTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSGSTSGSGKPGSGEGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFILTISSLEPEDFAVYYCQQRSNEPITFGQHTRLEIKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGISLEMIDSGIGDNLEAVDILGFDFTLGRFNNLRLIVERNNLYNITGFNINRINNVPIRFADESIWTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMERFVTVTAEALRFRQIQRGFRTTLDDLSGASYVNITAEDVDLTLNWGRESSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSH HHASAVAA SEQ IDexemplary MEIVETQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRL NO: 452cell-targeting LIYDASNRAIGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWmolecule 165 PITFGQGTRLEIKGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSIALQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGYFVTVSSEFPKPSTPPGSSGGAPGILGFVFTLKEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAI LGSVALILNSHHHASAVAASEQ ID exemplary MQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKP NO: 453cell-targeting WIYAPSNLASGVPARFSGSGSGFSYSLTISRVEAEDAATYYCQQWSFmolecule 166 NPPTFGAGTKLELKSGGGGSGGGGSGGGGSGGGGSGGGGSQAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFIDVWGTGTTVTVSEFPKPSTPPGSSGGILGFVFTLGAPKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVFGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTINWGRLSSVLPDYHGQDSVRVGRISFGSI NAILGSVALILNSHHHASAVAASEQ ID exemplary MQAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQ NO: 454cell-targeting GLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEmolecule 167 DSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSGSTSGSGKPGSGEGSQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGFSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKSEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISEGSINAILGSVALILNSH HHASAVAA SEQ IDexemplary ASVSDVPRDLEVVAATPTSLLISWCRQRCADSYRITYGETGGNSPV NO: 455cell-targeting QEFTVPGSWKTATISGLKPGVDYTITVYVVTHYYGWDRYSHPISINmolecule 168 YRTGSEFPKPSTPPGSSGGAPGILGFVFILKEFILDESTAKTYVDSLNVIRSAIGTPLQIISSGGISLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVITPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFWITLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID exemplaryMQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQ NO: 456 cell-targetingGLEWIGATYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSE molecule 169DSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSENVIRSAIGTPLQTISSGGTSLLMIDSGIGDNEFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTENWGRESSVLPDYHGQDSVRVGRISEGSINAILGSVALILNSHHHASAVAAGTGDALVALPEGESVRIADIVPGARPNSDNAIDLKVLDRHGNPVLADRLFHSGEHPVYTVRTVEGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAVIQRSAFSVDCAGFARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYYAKVASVTDAGVQPVYSLRVDTADHAFITNGFVSHATGLTGLNSGLTTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVPALWQLQ SEQ ID exemplaryMQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQ NO: 457 cell-targetingGLEWIGATYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSE molecule 170DSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSENVIRSAIGTPLQTISSGGTSLLMIDSGIGDNEFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTENWGRESSVLPDYHGQDSVRVGRISEGSINAILGSVALILNSHHHASAVAAGTGDALVALPEGESVRIADIVPGARPNSDNAIDLKVLDRHGNPVLADRLFHSGEHPVYTVRTVEGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAVIQRSAFSVDCAGFARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYYAKVASVTDAGVQPVYSLRVDTADHAFITNGFVSHATGLTGLNSGLTTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVPALWQLQ SEQ ID exemplaryMQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQ NO: 458 cell-targetingGLEWIGATYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSE molecule 171DSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSENVIRSAIGTPLQTISSGGTSLLMIDSGIGDNEFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTENWGRESSVLPDYHGQDSVRVGRISEGSINAILGSVALILN SHHHASAVAAKDEL SEQ IDexemplary MKEFTEDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 459cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 172 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKRSTPPGSSGGAPGILGFVFTLMQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQGLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEDSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISN PPTFGAGTKLELK SEQ IDexemplary MKEFTEDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 460cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 173 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIELTQSPSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPTFGGGTKLEIKGSTSGSGKPGSGEGSKVQLQESGPSLVQPSQRLSITCTVSGFSLISYGVHWVRQSPGKGLEWLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTAIYFCAKTLITTGYAMDYWGQGTTVT VSS SEQ ID exemplaryMKEFTEDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 461 cell-targetingNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 174DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIELTQSPSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPTFGGGTKLEIKGSTSGSGKPGSGEGSKVQLQESGPSLVQPSQRLSITCTVSGFSLISYGVHWVRQSPGKGLEWLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTAIYFCAKTLITTGYAMDYWGQGTTVT VSS SEQ ID exemplaryMKEFTEDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 462 cell-targetingNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 175DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIQMTQSPSSESASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDV WGQGTLVTVSS SEQ IDexemplary MKEFTEDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 463cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 176 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIQMTQSPSSESASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDV WGQGTLVTVSS SEQ IDexemplary MKEFTEDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 464cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 177 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIVMTQAAPSIPVTPGESVSISCRSSKSLLNSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGAGTKLELKGSTSGSGKPGSGEGSEVQLQQSGPELIKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGTYYYGSRVF DYWGQGTTLTVSS SEQ IDexemplary MKEFTEDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 465cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 178 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIVMTQAAPSIPVTPGESVSISCRSSKSLLNSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGAGTKLELKGSTSGSGKPGSGEGSEVQLQQSGPELIKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGTYYYGSRVF DYWGQGTTLTVSS SEQ IDexemplary MKEFTEDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 466cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 179 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIQLTQSPLSLPVTLGQPASISCRSSQSLVHRNGNTYLHWFQQRPGQSPRLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSSHVPPTFGAGTRLEIKGSTSGSGKPGSGEGSTKGQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGVNWIKQAPGQGLQWMGWINPNTGEPTFDDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVITCSRSRGKNEAW FAYWGQGTLVIVSS SEQ IDexemplary MKEFTEDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 467cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 180 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIQLTQSPLSLPVTLGQPASISCRSSQSLVHRNGNTYLHWFQQRPGQSPRLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSSHVPPTFGAGTRLEIKGSTSGSGKPGSGEGSTKGQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGVNWIKQAPGQGLQWMGWINPNTGEPTFDDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVITCSRSRGKNEAW FAYWGQGTLVIVSS SEQ IDexemplary MKEFTEDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 468cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 181 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAAHHSEDPSSKAPKAPGILGFVFTLEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTWADSVKGRFTISRDNAKNTWILQMNSLKPEDTAVYYCK RFRTAAQGTDYWGQGTQVTVSSSEQ ID exemplary MKEFTEDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNO: 469 cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 182 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAAHHSEDPSSKAPKAPGILGFVFTLEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTWADSVKGRFTISRDNAKNTWILQMNSLKPEDTAVYYCK RFRTAAQGTDYWGQGTQVTVSSSEQ ID exemplary MKEFTEDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNO: 470 cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 183 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAAHHSEDPSSKAPKAPGILGFVFTLEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTWADSVKGRFTISRDNAKNTWILQMNSLKPEDTAVYYCK RFRTAAQGTDYWGQGTQVTVSSSEQ ID exemplary MKEFTEDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNO: 471 cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 184 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAAHHSEDPSSKAPKAPGILGFVFTLEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTWADSVKGRFTISRDNAKNTWILQMNSLKPEDTAVYYCK RFRTAAQGTDYWGQGTQVTVSSSEQ ID exemplary MKEFTEDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNO: 472 cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 185 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIVLTQSPASLAVSLGQRATISCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPARFSGSGSGTDFSLTIHPVEEDDIAMYFCQQSRRVPYTFGGGTKLEIKGGGGSEVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYVNPFNDGTKYNEMFKGKATLTSDKSSSTAYMELSSLFSEDSAVYYCARQAWGYPWGQGTLVTVSA SEQ ID exemplaryMKEFTEDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 473 cell-targetingNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 186DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIVLTQSPASLAVSLGQRATISCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPARFSGSGSGTDFSLTIHPVEEDDIAMYFCQQSRRVPYTFGGGTKLEIKGGGGSEVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYVNPFNDGTKYNEMFKGKATLTSDKSSSTAYMELSSLFSEDSAVYYCARQAWGYPWGQGTLVTVSA SEQ ID exemplaryVTEHDTLLYKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 474 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 187NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID exemplaryGLDRNSGNYKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 475 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 188NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID exemplaryGVMTRGRLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 476 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 189NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID exemplaryVYALPLKMLKEFTLDFSTAKTYVDSLNVIRSAIGFPLQTISSGGTSLL NO: 477 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 190NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID exemplaryNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 478 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 191NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQ GSLVTVSS SEQ ID exemplaryGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLM NO: 479 cell-targetingIDSGIGDNLFAVDILGFIWILGRFNNLRLIVERNNLYVTGFVNRTNN molecule 192VFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQ GSLVTVSS SEQ ID exemplaryDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKL NO: 480 cell-targetingLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTT molecule 193PPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSSEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHH HASAVAA SEQ ID exemplaryQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKWPGQG NO: 481 cell-targetingLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEDS molecule 194AVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGIPLQTISSGGTSLLMIDSGIGDNLFAVDILGIFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSH HHASAVAA SEQ IDexemplary KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 482cell-targeting FAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSmolecule 195 HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSVTEHDTLLY SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 483 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 196HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSNLVPMVATV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 484 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 197HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSQYDPVAALF SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 485 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 198HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSCLGGLLTMV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 486 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 199HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSILRGSVAHK SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 487 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 200HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHAVVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSN LVPMVATV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 488 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 201HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARLYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSNLVPMVATV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 489 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 202HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQ GTKVEIKGILGFVFTL SEQ IDexemplary KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 490cell-targeting FAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSmolecule 203 HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDVHWVRQAPGQRLEWMGWLHADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARERIQLWFDYWGQGTLVTVSSNLVPMVATV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 491 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 204HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSNLVPMVATV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 492 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 205HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQGLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEDSAVYYCARSNYYGSSYVWFFDVWGAGITVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELK NLVPMVATV SEQ IDexemplary KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 493cell-targeting FAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSmolecule 206 HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARAQLRPNYWYFDVWGACTTVTVSSGGGGSDIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKNLVPMVATV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 494 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 207HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAGSTSGSGKPGSGEGSTKGQIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEI KNLVPMVATV SEQ IDexemplary KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 495cell-targeting FAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSmolecule 208 HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRERTAAQGTD YWGQGTQVTVSSAHHSEDNLVPVATVSEQ ID exemplary KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLNO: 496 cell-targeting FAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSmolecule 209 HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIELTQSPSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPTFGGGTKLEIKGSTSGSGKPGSGEGSKVQLQESGPSLVQPSQRLSITCTVSGFSLISYGVHWVRQSPGKGLEWLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTAIYFCAKTLITTYAMDYWGQGTTVTVSSNLVPMVA TV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 497 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 210HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAAHHSEDPSSKAPKAPEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTD YWGQGTQVTVSSNLVPMVATVSEQ ID exemplary KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLNO: 498 cell-targeting FAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSmolecule 211 HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPASVSDVPRDLEVVAATPTSLLISWCRQRCADSYRITYGETGGNSPVQEFTVPGSWKTATISGLKPGVDYTITVYVVTHYYGWDRYSHPISINYRTGSNLVPMVATV SEQ ID exemplaryASVSDVPRDLEVVAATPTSLLISWCRQRCADSYRITYGETGGNSPV NO: 499 cell-targetingQEFTVPGSWKTATISGLKPGVDYTITVYVVTHYYHWDRYSHPISIN molecule 212YRTGSEFPKPSTPPGSSGGAPKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISEGSINAILGSVALILNSHHHASAVAAANLVPMVA TV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 500 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 213HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIVLTQSPASLAVSLGQRATISCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPARFSGSGSGTDFSLTIHPVEEDDIAMYFCQQSRRVPYTFGGGTKLEIKGGGGSEVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYVNPFNDGTKYNEMFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARQAWGYPWGGTLVTVSANLVPMVATV SEQ ID exemplaryNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 501 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 214NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAGGGGSGGDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID exemplaryNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 502 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 215NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRESGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQ GSLVTVSS SEQ ID exemplaryNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 503 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 216NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS SEQ ID exemplaryGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLM NO: 504 cell-targetingIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNN molecule 217VFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWHHWVRQAPGKGLEWVAWISPYGGSTYYNDSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQY LYHPATFGQGTKVEIK SEQ IDexemplary NLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 505cell-targeting MIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNmolecule 218 NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAGGGGSGGDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAFYYCQQYNSYPYTFGQGTKLEIKGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDVHWVRQAPGQRLEWMGWLHADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARERIQLWFDYWGQGTLVTVSS SEQ ID exemplaryNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 506 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 219NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID exemplaryNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 507 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 220NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNNHWVKQTPGQGLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEDSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFG AGTKLELK SEQ IDexemplary NLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 508cell-targeting MIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNmolecule 221 NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVAIILNSHHHASAVAAGGGGSGGQVQLVQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARAQLRPNYWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNP PTFGAGTKLELK SEQ IDexemplary APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYM NO: 509cell-targeting PKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLmolecule 222 ELKGSETTFMCEYADETATIVEFLNRWITFCQSHSTLTEFPKPSTPPGSSGGAPNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGITAVTLSADSSYTTLQRVAGISRTGMQNIRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID exemplaryGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLM NO: 510 cell-targetingIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNN molecule 223VFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSHSTLT SEQ ID exemplaryMNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTS NO: 511 cell-targetingLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNR molecule 224TNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSITSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMTTGFVMDSWG QGSLVTVSS SEQ ID exemplaryMNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTS NO: 512 cell-targetingLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNR molecule 225TNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSITSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMTTGFVMDSWG QGSLVTVSS SEQ ID exemplaryMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 513 cell-targetingNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 226DFSHVTFPGTTAVTLSADSSYTTLQRSVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVS SNLVPMVATV SEQ IDexemplary MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 514cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 227 DFSHVTFPGTTAVTLSADSSYTTLQRSVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVS SNLVPMVATV SEQ IDexemplary MGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 515cell-targeting MIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGVNRTNmolecule 228 NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFYMDSWGQ GSLVTVSS SEQ ID exemplaryMDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAP NO: 516 cell-targetingKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHY molecule 229TTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGGNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSSEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSH HHASAVAA SEQ IDexemplary MQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQ NO: 517cell-targeting GLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEmolecule 230 DSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN SHHHASAVAA SEQ IDexemplary MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 518cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 231 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSNLVPMVATV SEQ ID exemplaryMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 519 cell-targetingNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 232DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATF GQGTKVEIKGILGFVFTLSEQ ID exemplary MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNO: 520 cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 233 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATF GQGTKVEIKGILGFVFTLSEQ ID exemplary MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNO: 521 cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 234 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDVHWVRQAPGQRLEWMGWLHADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARERIQLWFDYWGQGTLVTVSSNLVPMVATV SEQ ID exemplaryMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 522 cell-targetingNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 235DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSNLVPMVATV SEQ ID exemplaryQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQG NO: 523 cell-targetingLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEDS molecule 236AVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSH HHASAVAA SEQ IDexemplary MQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQ NO: 524cell-targeting GLEWIGAIYTGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEmolecule 237 DSAVYYCARSNYYGSSYVWFFDWYGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGFSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN SHHHASAVAA SEQ IDexemplary MDIQMIQSPSSLSASVGDRVIITCRASQDVNTAVAWYQQKPGKAP NO: 525cell-targeting KLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYmolecule 238 TTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDITAVYYCSRWGGDGFYAMDVWGQGYLVTVSSEFPKPSYPPGSSGGAPGILHFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSH HHASAVAAKDEL SEQ IDexemplary MDIQMIQSPSSLSASVGDRVIITCRASQDVNTAVAWYQQKPGKAP NO: 526cell-targeting KLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYmolecule 239 TTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDITAVYYCSRWGGDGFYAMDVWGQGYLVTVSSEFPKPSYPPGSSGGAPGILHFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSH HHASAVAAKDEL SEQ IDexemplary MDIELTQSPSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGNAPRL NO: 527cell-targeting LISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSmolecule 240 TPTFGGGIKLEIKGSTSGSGKPGSGEGSKVQLQESGPSLVQPSQRLSITCTVSGFSLISYGVHWVRQSPGKGLEWLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTAIYFCAKTLITTGYAMDYWGQGTTVTVSSEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNIFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAV AAKDEL SEQ ID exemplaryMDIELTQSPSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGNAPRL NO: 528 cell-targetingLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWS molecule 241TPTFGGGIKLEIKGSTSGSGKPGSGEGSKVQLQESGPSLVQPSQRLSITCTVSGFSLISYGVHWVRQSPGKGLEWLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTAIYFCAKTLITTGYAMDYWGQGTTVTVSSEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNIFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAV AAKDEL SEQ ID exemplaryMDIVMTQAAPSIPVTPGESVSISCRSSKSLLNSNGNTYLYWFLQRPG NO: 529 cell-targetingQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYC molecule 242MQHLEYPFTFGAGTKLELKGSTSGSGKPGSGEGSEVQLQQSGPELIKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTTAYMELSSLTSEDSAVYYCARGTYYYGSRVFDYWGQGTTLTVSSAEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDKTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSV ALILNSHHHASAVAAKDELSEQ ID exemplary MDIVMTQAAPSIPVTPGESVSISCRSSKSLLNSNGNTYLYWFLQRPG NO: 530cell-targeting QSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCmolecule 243 MQHLEYPFTFGAGTKLELKGSTSGSGKPGSGEGSEVQLQQSGPELIKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTTAYMELSSLTSEDSAVYYCARGTYYYGSRVFDYWGQGTTLTVSSAEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDKTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSV ALILNSHHHASAVAAKDELSEQ ID exemplary MDIQLTQSPLSLPVTLGQPASISCRSSQSLVHRNGNTYLHWFQQRPG NO: 531cell-targeting QSPRLLIYTVSNRFSGVPDRESGSGSGTDFTLKISRVEAEDVGVYFCSmolecule 244 QSSHVPPTFGAGTRLEIKGSTSGSGKPGSGEGSTKGQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGVNWIKQAPGQGLQWMGWINPNTGEPTFDDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCSRSRGKNEAWFAYWGQGTLVTVSSEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALI LNSHHHASAVAAKDEL SEQ IDexemplary MDIQLTQSPLSLPVTLGQPASISCRSSQSLVHRNGNTYLHWFQQRPG NO: 532cell-targeting QSPRLLIYTVSNRFSGVPDRESGSGSGTDFTLKISRVEAEDVGVYFCSmolecule 245 QSSHVPPTFGAGTRLEIKGSTSGSGKPGSGEGSTKGQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGVNWIKQAPGQGLQWMGWINPNTGEPTFDDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCSRSRGKNEAWFAYWGQGTLVTVSSEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALI LNSHHHASAVAAKDEL SEQ IDexemplary MEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQ NO: 533cell-targeting RELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAmolecule 246 VYYCKRFRTAAQGTDYWGQGTQVTVSSAHHSEDPSSKAPKAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAKDEL SEQ ID exemplaryMEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQ NO: 534 cell-targetingRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA molecule 247VYYCKRFRTAAQGTDYWGQGTQVTVSSAHHSEDPSSKAPKAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAKDEL SEQ ID exemplaryMEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQ NO: 535 cell-targetingRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA molecule 248VYYCKRFRTAAQGTDYWGQGTQVTVSSAHHSEDPSSKAPKAPGILGFVFTLGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAKDEL SEQ ID exemplaryMEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQ NO: 536 cell-targetingRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA molecule 249VYYCKRFRTAAQGTDYWGQGTQVTVSSEFPKPSTPPGSSGGAPGILGFVFTLGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAKDEL SEQ ID exemplaryMAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFY NO: 537 cell-targetingMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVI molecule 250VLELKGSETTFMCEYADETATIVEFLNRWITFCQSHSTLTEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAKDEL SEQ ID exemplaryMAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFY NO: 538 cell-targetingMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVI molecule 251VLELKGSETTFMCEYADETATIVEFLNRWITFCQSHSTLTEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAKDEL SEQ ID exemplaryMQVQLVQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQ NO: 539 cell-targetingGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSE molecule 252DSAVYYCARAQLRPNYWYFDVWGAGTTVTVSSGSTSGSGKPGSGEGSDIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSH HHASAVAA SEQ IDexemplary MQVQLVQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQ NO: 540cell-targeting GLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEmolecule 253 DSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN SHHHASAVAA SEQ IDexemplary MQVQLVQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQ NO: 541cell-targeting GLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEmolecule 254 DSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN SHHHASAVAA SEQ IDexemplary MQVQLVQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQ NO: 542cell-targeting GLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEmolecule 255 DSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN SHHHASAVAA SEQ IDexemplary MQVQLQOPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGR NO: 543cell-targeting GLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEmolecule 256 DSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAGSTSGSGKPGSGEGSTKGQIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN SHHHASAVAA SEQ IDexemplary MEVQLNESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGK NO: 544cell-targeting GLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEmolecule 257 DTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSGSTSGSGKPGSGEGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSH HHASAVAA SEQ IDexemplary MEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRL NO: 545cell-targeting LIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWmolecule 258 PITFGQGTRLEIKGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRIGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAI LGSVALILNSHHHASAVAASEQ ID exemplary MQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKP NO: 546cell-targeting WIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFmolecule 259 NPPTFGAGTKLELKSGGGGSGGGGSGGGGSGGGGSGGGGSQAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSEFPKPSTPPGSSGGILGFVFTLGAPKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFTVTVAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSI NAILGSVALILNSHHHASAVAASEQ ID exemplary MQAYLQQSGAELVRPGASVKMSCKASCWTFTSYNMHWVKQTPRQ NO: 547cell-targeting GLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEmolecule 260 DSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSGSTSGSGKPGSGEGSQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKSEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSH HHASAVAA SEQ IDexemplary ASVSDVPRDLEVVAATPTSLLISWCRQRCADSYRITYGETGGNSPV NO: 548cell-targeting QEFTVPGSWKTATISGLKPGVDYTITVYVVTHYYGWDRYSHPISINmolecule 261 YRTGSEFPKPSTPPGSSGGAPGILGFVTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA NLVPMVATV SEQ IDexemplary MQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQ NO: 549cell-targeting GLEWIGAIYPGNGDTSFNQKFKGRATLTADKSSSTVYMQLSSLTSEmolecule 262 DSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEEPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN SHHHASAVAAKDEL SEQ IDexemplary MKEFTLDGSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 550cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 263 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLMQVQLQQPGAEINKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQGLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEDSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISN PPTFGAGTKLELK SEQ IDexemplary MKEFTLDGSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 551cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 264 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIELTQSPSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPTFGGGTKLEIKGSTSGSGKPGSGEGSKVQLQESGPSLVQPRSQRLSITCTVSGFSLISYGVHWVRQSPGKGLEWLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTAIYFCAKTLITTGYAMDYWGQGTTVT VSS SEQ ID exemplaryMKEFTLDGSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 552 cell-targetingNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 265DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIELTQSPSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPTFGGGTKLEIKGSTSGSGKPGSGEGSKVQLQESGPSLVQPRSQRLSITCTVSGFSLISYGVHWVRQSPGKGLEWLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTAIYFCAKTLITTGYAMDYWGQGTTVT VSS SEQ ID exemplaryMKEFTLDGSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 553 cell-targetingNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 266DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDV WGQGTLVTVSS SEQ IDexemplary MKEFTLDGSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 554cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 267 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDV WGQGTLVTVSS SEQ IDexemplary MKEFTLDGSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 555cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 268 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIVMTQAAPSIPVTPGESVSISCRSSKSLLNSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGAGTKLELKGSTSGSGKPGSGEGSEVQLQQSGPELIKPGASVKMSCKASCWTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGTYYYGSRVF DYWGQGTTLTVSS SEQ IDexemplary MKEFTLDGSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 556cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 269 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIVMTQAAPSIPVTPGESVSISCRSSKSLLNSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGAGTKLELKGSTSGSGKPGSGEGSEVQLQQSGPELIKPGASVKMSCKASCWTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGTYYYGSRVF DYWGQGTTLTVSS SEQ IDexemplary MKEFTLDGSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 557cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 270 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIQLTQSPLSLPVTLGQPASISCRSSQSLVHRNGNTYLHWFQQRPGQSPRLLIYTVSNRFGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSSHVPPTFGAGTRLEIKGSTSGSGKPGSGEGSTKGQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGVNWIKQAPGQGLQWMGWINPNTGEPTFDDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCSRSRGKNEAW FAYWGQGTLVTVTSS SEQ IDexemplary MKEFTLDGSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 558cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 271 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIQLTQSPLSLPVTLGQPASISCRSSQSLVHRNGNTYLHWFQQRPGQSPRLLIYTVSNRFGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSSHVPPTFGAGTRLEIKGSTSGSGKPGSGEGSTKGQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGVNWIKQAPGQGLQWMGWINPNTGEPTFDDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCSRSRGKNEAW FAYWGQGTLVTVTSS SEQ IDexemplary MKEFTLDGSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 559cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 272 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAAHHSEDPSSKAPKAPGILGFVFTLEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCK RFRTAAQGTDYWGQGTQVTVSSSEQ ID exemplary MKEFTLDGSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNO: 560 cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 273 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAAHHSEDPSSKAPKAPGILGFVFTLEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCK RFRTAAQGTDYWGQGTQVTVSSSEQ ID exemplary MKEFTLDGSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNO: 561 cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 274 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAAHHSEDPSSKAPKAPGILGFVFTLEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCK RFRTAAQGTDYWGQGTQVTVSSSEQ ID exemplary MKEFTLDGSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNO: 562 cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 275 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAAHHSEDPSSKAPKAPGILGFVFTLEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCK RFRTAAQGTDYWGQGTQVTVSSSEQ ID exemplary MKEFTLDGSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNO: 563 cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 276 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLASVSDVPRDLEVVAATPTSLLISWCRQRCADSYRITYGETGGNSPVQEFTVPGSWKTATISGLKPGVDYTITVYVVTHYYGWDRYSHPISINYRTGS SEQ ID exemplaryMKEFTLDGSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 564 cell-targetingNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 277DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIVLTQSPASLAVSLGQRATISCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPARFSGSGSGTDFSLTIHPVEEDDIAMYFCQQSRRVPYTFGGGTKLEIKGGGGSEVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYVNPFNDGTKYNEMFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARQAWGYPWGQGTLVTVSA SEQ ID exemplaryVTEHDTLLYKEFTLDFSTAKTYYDSLNVIRSAIGTPLQTISSGGTSLL NO: 565 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 278NAFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLYWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID exemplaryGLDRNSGNYKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 566 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 279NAFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLYWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID exemplaryGVMTRGRLKEFTLDFSTAKTYNDSLNVIRSAIGTPLQTISSGGTSLL NO: 567 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 280NAFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLYWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID exemplaryVYALPLKMLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 568 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 281NAFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLYWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID exemplaryNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 569 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 282NAFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLYWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQ GSLVTVSS SEQ ID exemplaryGILGFVFTLAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLM NO: 570 cell-targetingIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNN molecule 283AFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLYWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQ GSLVTVSS SEQ ID exemplaryDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKL NO: 571 cell-targetingLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTT molecule 284PPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSSEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNAFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRIQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHH HASAVAA SEQ ID exemplaryQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQG NO: 572 cell-targetingLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEDS molecule 285AVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARGSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNAFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSH HHASAVAA SEQ IDexemplary KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 573cell-targeting FAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNAFYRFADFSmolecule 286 HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSVTEHDTLLY SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 574 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNAFYRFADFS molecule 287HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSNLVPMVATV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 575 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNAFYRFADFS molecule 288HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGLVTVSSQYDPVAALF SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 576 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNAFYRFADFS molecule 289HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSCLGGLLTMV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 577 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNAFYRFADFS molecule 290HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSILRGSVAHK SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 578 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNAFYRFADFS molecule 291HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHAVVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMFTTGFVMDSWGQGSLVTVSSN LVPMVATV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 579 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNAFYRFADFS molecule 292HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIIIWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSNLVPMVATV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 580 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNAFYRFADFS molecule 293HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQ GTKVEIKGILGFVFTL SEQ IDexemplary KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 581cell-targeting FAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNAFYRFADFSmolecule 294 HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDVHWVRQAPGQRLEWMGWLHADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARERIQLWFDYWGQGTLVTVSSNLVPMVATV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 582 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNAFYRFADFS molecule 295HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSNLVPMVATV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 583 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNAFYRFADFS molecule 296HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQGLEWIGAIYPGNGDTSFNIQKFKGKATLTADKSSSTVYMQLSSLTSEDSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELK NLVPMVATV SEQ IDexemplary KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 584cell-targeting FAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNAFYRFADFSmolecule 297 HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARAQLRPNYWYFDVWGAGTTVTVSSGGGGSDIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKNLVPMVATV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 585 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNAFYRFADFS molecule 298HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAGSTSGSGKPGSGEGSTKGQIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQWTSNPPTFGGGTKLEI KNLVPMVATV SEQ IDexemplary KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 586cell-targeting FAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNAFYRFADFSmolecule 299 HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTDYWGQGTQVTVSSAHHSEDNLVPMVATV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 587 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNAFYRFADFS molecule 300HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQADRVTITCKASEDIYNRLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPTFGGGTKLEIKGSTSGSGKPGSGEGSKVQLQESGPSLVQPSQRLSITCTVSGFSLISYGVHWVRQSPGKGLEWLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTAIYFCAKTLITTGYAMDYWGQGTTVTVSSNLVPMVA TV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 588 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNAFYRFADFS molecule 301HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAAHHSEDPSSKAPKAPEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTD YWGQGTQVTVSSNLVPMVATVSEQ ID exemplary KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLNO: 589 cell-targeting FAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNAFYRFADFSmolecule 302 HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIVLTQSPASLAVSLGQRATISCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPARFSGSGSGTDFSLTIHPVEEDDIAMYFCQQSRRVPYFFGGGFKLEIKGGGGSEVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYVNPFNDGTKYNEMFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARQAWGYPWGQGTLVTVSANLVPMVATV SEQ ID exemplaryASVSDVPRDLEVVAATPTSLLISWCRQRCADSYRITYGETGGNSPV NO: 590 cell-targetingQEFTVPGSWKTATISGLKPGVDYTITVYVVTHYYGWDRYSHPISIN molecule 303YRTGSEFPKPSTPPGSSGGAPKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGEDFTLGRFNNLRLIVERNNLYVTGFVNRTNNAFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMYLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAANLVPMVA TVNLVPMVATV SEQ IDexemplary KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 591cell-targeting FAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNAFYRFADFSmolecule 304 HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIVLTQSPASLAVSLGQRATISCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPARFSGSGSGTDFSLTIHPVEEDDIAMYFCQQSRRVPYFFGGGFKLEIKGGGGSEVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYVNPFNDGTKYNEMFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARQAWGYPWGQGTLVTVSANLVPMVATV SEQ ID exemplaryNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 592 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 305NAFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAGGGGSGGDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQNSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID exemplaryNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 593 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 306NAFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQ GSLVTVSS SEQ ID exemplaryNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 594 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 307NAFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIIIWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSERAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS SEQ ID exemplaryGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTTSLLM NO: 595cell-targeting IDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNmolecule 308 AFYREADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISSGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQY LYHPATFGQGTKVEIK SEQ IDexemplary NLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 596cell-targeting MIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNmolecule 309 NAFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAGGGGSGGDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVTSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDVHWVRQAPGQRLEWMGWLHADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARERIQLWFDYWGQGTLVTVSS SEQ ID exemplaryNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 597 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 310NAFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID exemplaryNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 598 cell-targetingMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTN molecule 311NAFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQGLEWVIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEDSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFG AGTKLELK SEQ IDexemplary NLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 599cell-targeting MIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNmolecule 312 NAFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAGGGGSGGQVQLVQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARAQLRPNYWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIVLSQSPAILSASPGEKVTMTGRASSSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNP PTFGAGTKLELK SEQ IDexemplary APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYM NO: 600cell-targeting PKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLmolecule 313 ELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTEFPKPSTPPGSSGGAPNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNAFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID exemplaryGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTTSLLM NO: 601cell-targeting IDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNmolecule 314 AFYREADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT SEQ ID exemplaryMNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTS NO: 602 cell-targetingLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNR molecule 315TNNAFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWG QGSLVTVSS SEQ ID exemplaryMNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTS NO: 603 cell-targetingLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNR molecule 316TNNAFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWG QGSLVTVSS SEQ ID exemplaryMKEFTLDGSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 604 cell-targetingNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 317DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLYWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQYQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGOGSLVTVS SNLVPMVATV SEQ IDexemplary MKEFTLDGSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 605cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 318 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLYWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQYQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGOGSLVTVS SNLVPMVATV SEQ IDexemplary MGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 606cell-targeting MIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNmolecule 319 NVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQ GSLVTVSS SEQ ID exemplaryMDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAP NO: 607 cell-targetingKLLIYSASFLYSGVPSRFSGSRSGTDFILTISSLQPEDFATYYCQQHY molecule 320TTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMINSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSSEFPKTSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTISYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSH HHASAVAA SEQ IDexemplary MQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQ NO: 608cell-targeting GLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEmolecule 321 DSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN SHHHASAVAA SEQ IDexemplary MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 609cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 322 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEIWDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVWRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSNLVPMVATV SEQ ID exemplaryMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 610 cell-targetingNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 323DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATF GQGTKVEIKGILGFVFTLSEQ ID exemplary MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNO: 611 cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 324 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATF GQGTKVEIKGILGFVFTLSEQ ID exemplary MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNO: 612 cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 325 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDVHWVRQAPGQRLEWMGWLHADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARERIQLWFDYWGQGTLVTVSSNLVPMVATV SEQ ID exemplaryMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 613 cell-targetingNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 326DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSNLVPMVATV SEQ ID exemplaryQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQG NO: 614 cell-targetingLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEDS molecule 327AVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSH HHASAVAA SEQ IDexemplary MQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQ NO: 615cell-targeting GLEWIGAIYTGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEmolecule 328 DSAVYYCARSNYYGSSYVWFFDWYGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGFSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN SHHHASAVAA SEQ IDexemplary MDIQMIQSPSSLSASVGDRVIITCRASQDVNTAVAWYQQKPGKAP NO: 616cell-targeting KLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYmolecule 329 TTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDITAVYYCSRWGGDGFYAMDVWGQGYLVTVSSEFPKPSYPPGSSGGAPGILHFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSH HHASAVAAKDEL SEQ IDexemplary MDIQMIQSPSSLSASVGDRVIITCRASQDVNTAVAWYQQKPGKAP NO: 617cell-targeting KLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYmolecule 330 TTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDITAVYYCSRWGGDGFYAMDVWGQGYLVTVSSEFPKPSYPPGSSGGAPGILHFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSH HHASAVAAKDEL SEQ IDexemplary MDIELTQSPSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGNAPRL NO: 618cell-targeting LISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSmolecule 331 TPTFGGGIKLEIKGSTSGSGKPGSGEGSKVQLQESGPSLVQPSQRLSITCTVSGFSLISYGVHWVRQSPGKGLEWLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTAIYFCAKTLITTGYAMDYWGQGTTVTVSSEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNIFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAV AAKDEL SEQ ID exemplaryMDIELTQSPSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGNAPRL NO: 619 cell-targetingLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWS molecule 332TPTFGGGIKLEIKGSTSGSGKPGSGEGSKVQLQESGPSLVQPSQRLSITCTVSGFSLISYGVHWVRQSPGKGLEWLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTAIYFCAKTLITTGYAMDYWGQGTTVTVSSEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNIFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAV AAKDEL SEQ ID exemplaryMDIVMTQAAPSIPVTPGESVSISCRSSKSLLNSNGNTYLYWFLQRPG NO: 620 cell-targetingQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYC molecule 333MQHLEYPFTFGAGTKLELKGSTSGSGKPGSGEGSEVQLQQSGPELIKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTTAYMELSSLTSEDSAVYYCARGTYYYGSRVFDYWGQGTTLTVSSAEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDKTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSV ALILNSHHHASAVAAKDELSEQ ID exemplary MDIVMTQAAPSIPVTPGESVSISCRSSKSLLNSNGNTYLYWFLQRPG NO: 621cell-targeting QSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCmolecule 334 MQHLEYPFTFGAGTKLELKGSTSGSGKPGSGEGSEVQLQQSGPELIKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTTAYMELSSLTSEDSAVYYCARGTYYYGSRVFDYWGQGTTLTVSSAEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDKTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSV ALILNSHHHASAVAAKDELSEQ ID exemplary MDIQLTQSPLSLPVTLGQPASISCRSSQSLVHRNGNTYLHWFQQRPG NO: 622cell-targeting QSPRLLIYTVSNRFSGVPDRESGSGSGTDFTLKISRVEAEDVGVYFCSmolecule 335 QSSHVPPTFGAGTRLEIKGSTSGSGKPGSGEGSTKGQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGVNWIKQAPGQGLQWMGWINPNTGEPTFDDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCSRSRGKNEAWFAYWGQGTLVTVSSEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALI LNSHHHASAVAAKDEL SEQ IDexemplary MDIQLTQSPLSLPVTLGQPASISCRSSQSLVHRNGNTYLHWFQQRPG NO: 623cell-targeting QSPRLLIYTVSNRFSGVPDRESGSGSGTDFTLKISRVEAEDVGVYFCSmolecule 336 QSSHVPPTFGAGTRLEIKGSTSGSGKPGSGEGSTKGQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGVNWIKQAPGQGLQWMGWINPNTGEPTFDDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCSRSRGKNEAWFAYWGQGTLVTVSSEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALI LNSHHHASAVAAKDEL SEQ IDexemplary MEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQ NO: 624cell-targeting RELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAmolecule 337 VYYCKRFRTAAQGTDYWGQGTQVTVSSAHHSEDPSSKAPKAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNAFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAKDEL SEQ ID exemplaryMEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQ NO: 625 cell-targetingRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA molecule 338VYYCKRFRTAAQGTDYWGQGTQVTVSSAHHSEDPSSKAPKAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNAFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAKDEL SEQ ID exemplaryMEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQ NO: 626 cell-targetingRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA molecule 339VYYCKRFRTAAQGTDYWGQGTQVTVSSAHHSEDPSSKAPKAPGILGFVFTLGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAKDEL SEQ ID exemplaryMEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQ NO: 627 cell-targetingRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA molecule 340VYYCKRFRTAAQGTDYWGQGTQVTVSSEFPKPSTPPGSSGGAPGILGFVFTLGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAKDEL SEQ ID exemplaryMAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFY NO: 628 cell-targetingMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVI molecule 341VLELKGSETTFMCEYADETATIVEFLNRWITFCQSHSTLTEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAKDEL SEQ ID exemplaryMAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFY NO: 629 cell-targetingMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVI molecule 342VLELKGSETTFMCEYADETATIVEFLNRWITFCQSHSTLTEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAKDEL SEQ ID exemplaryMQVQLVQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQ NO: 630 cell-targetingGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSE molecule 343DSAVYYCARAQLRPNYWYFDVWGAGTTVTVSSGSTSGSGKPGSGEGSDIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSH HHASAVAA SEQ IDexemplary MQVQLVQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQ NO: 631cell-targeting GLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEmolecule 344 DSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN SHHHASAVAA SEQ IDexemplary MQVQLVQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQ NO: 632cell-targeting GLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEmolecule 345 DSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN SHHHASAVAA SEQ IDexemplary MQVQLVQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQ NO: 633cell-targeting GLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEmolecule 346 DSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN SHHHASAVAA SEQ IDexemplary MQVQLQOPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGR NO: 634cell-targeting GLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEmolecule 347 DSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAGSTSGSGKPGSGEGSTKGQIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN SHHHASAVAA SEQ IDexemplary MEVQLNESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGK NO: 635cell-targeting GLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEmolecule 348 DTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSGSTSGSGKPGSGEGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSH HHASAVAA SEQ IDexemplary MEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRL NO: 636cell-targeting LIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWmolecule 349 PITFGQGTRLEIKGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRIGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAI LGSVALILNSHHHASAVAASEQ ID exemplary MQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKP NO: 637cell-targeting WIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFmolecule 350 NPPTFGAGTKLELKSGGGGSGGGGSGGGGSGGGGSGGGGSQAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSEFPKPSTPPGSSGGILGFVFTLGAPKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNAFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFTVTVAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSI NAILGSVALILNSHHHASAVAASEQ ID exemplary MQAYLQQSGAELVRPGASVKMSCKASCWTFTSYNMHWVKQTPRQ NO: 638cell-targeting GLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEmolecule 351 DSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSGSTSGSGKPGSGEGSQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKSEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSH HHASAVAA SEQ IDexemplary ASVSDVPRDLEVVAATPTSLLISWCRQRCADSYRITYGETGGNSPV NO: 639cell-targeting QEFTVPGSWKTATISGLKPGVDYTITVYVVTHYYGWDRYSHPISINmolecule 352 YRTGSEFPKPSTPPGSSGGAPGILGFVTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA NLVPMVATV SEQ IDexemplary MQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQ NO: 640cell-targeting GLEWIGAIYPGNGDTSFNQKFKGRATLTADKSSSTVYMQLSSLTSEmolecule 353 DSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEEPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN SHHHASAVAAKDEL SEQ IDexemplary MKEFTLDGSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 641cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 354 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLMQVQLQQPGAEINKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQGLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEDSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISN PPTFGAGTKLELK SEQ IDexemplary MKEFTLDGSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 642cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 355 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIELTQSPSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPTFGGGTKLEIKGSTSGSGKPGSGEGSKVQLQESGPSLVQPRSQRLSITCTVSGFSLISYGVHWVRQSPGKGLEWLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTAIYFCAKTLITTGYAMDYWGQGTTVT VSS SEQ ID exemplaryMKEFTLDGSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 643 cell-targetingNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 356DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIELTQSPSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPTFGGGTKLEIKGSTSGSGKPGSGEGSKVQLQESGPSLVQPRSQRLSITCTVSGFSLISYGVHWVRQSPGKGLEWLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTAIYFCAKTLITTGYAMDYWGQGTTVT VSS SEQ ID exemplaryMKEFTLDGSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 644 cell-targetingNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 357DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDV WGQGTLVTVSS SEQ IDexemplary MKEFTLDGSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 645cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 358 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDV WGQGTLVTVSS SEQ IDexemplary MKEFTLDGSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 646cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 359 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIVMTQAAPSIPVTPGESVSISCRSSKSLLNSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGAGTKLELKGSTSGSGKPGSGEGSEVQLQQSGPELIKPGASVKMSCKASCWTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGTYYYGSRVF DYWGQGTTLTVSS SEQ IDexemplary MKEFTLDGSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 647cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 360 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIVMTQAAPSIPVTPGESVSISCRSSKSLLNSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGAGTKLELKGSTSGSGKPGSGEGSEVQLQQSGPELIKPGASVKMSCKASCWTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGTYYYGSRVF DYWGQGTTLTVSS SEQ IDexemplary MKEFTLDGSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 648cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 361 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIQLTQSPLSLPVTLGQPASISCRSSQSLVHRNGNTYLHWFQQRPGQSPRLLIYTVSNRFGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSSHVPPTFGAGTRLEIKGSTSGSGKPGSGEGSTKGQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGVNWIKQAPGQGLQWMGWINPNTGEPTFDDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCSRSRGKNEAW FAYWGQGTLVTVTSS SEQ IDexemplary MKEFTLDGSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 649cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 362 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIQLTQSPLSLPVTLGQPASISCRSSQSLVHRNGNTYLHWFQQRPGQSPRLLIYTVSNRFGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSSHVPPTFGAGTRLEIKGSTSGSGKPGSGEGSTKGQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGVNWIKQAPGQGLQWMGWINPNTGEPTFDDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCSRSRGKNEAW FAYWGQGTLVTVTSS SEQ IDexemplary MKEFTLDGSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 650cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 363 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAAHHSEDPSSKAPKAPGILGFVFTLEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCK RFRTAAQGTDYWGQGTQVTVSSSEQ ID exemplary MKEFTLDGSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNO: 651 cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 364 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAAHHSEDPSSKAPKAPGILGFVFTLEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCK RFRTAAQGTDYWGQGTQVTVSSSEQ ID exemplary MKEFTLDGSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNO: 652 cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 365 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAAHHSEDPSSKAPKAPGILGFVFTLEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCK RFRTAAQGTDYWGQGTQVTVSSSEQ ID exemplary MKEFTLDGSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNO: 653 cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 366 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAAHHSEDPSSKAPKAPGILGFVFTLEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCK RFRTAAQGTDYWGQGTQVTVSSSEQ ID exemplary MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNO: 654 cell-targeting NLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNAFYRFAmolecule 367 DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIVLTQSPASLAVSLGQRATISCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPARFSGSGSGTDFSLTIHMTEEDDIAMYFCQQSRRVPYTFGGGTKLEIKGGGGSEVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYVNPFNDGTKYNEMFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARQAWGYPWGQGTLVTVSA SEQ ID exemplaryMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGD NO: 655 cell-targetingNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNAFYRFA molecule 368DFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIVLTQSPASLAVSLGQRATISCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPARFSGSGSGTDFSLTIHMTEEDDIAMYFCQQSRRVPYTFGGGTKLEIKGGGGSEVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYVNPFNDGTKYNEMFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARQAWGYPWGQGTLVTVSA SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNL NO: 656 cell-targetingFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS molecule 369HVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIVLTQSPASLAVSLGQRATISCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPARFSGSGSGTDFSLTIHPVEEDDIAMYFCQQSRRVPYFFGGGFKLEIKGGGGSEVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYVNPFNDGTKYNEMFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARQAWGYPWGQGTLVTVSANLVPMVATV SEQ ID exemplaryVTEHDTLLYKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 657 cell-targetingMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRT molecule 370NNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYATMTAEDVDLTLNWGRLSSVLPDYFIGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFKSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID exemplaryGLDRNSGNYKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 658 cell-targetingMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRT molecule 371NNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYATMTAEDVDLTLNWGRLSSVLPDYFIGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFKSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID exemplaryGVMTRGRLKEFTLDESTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 659 cell-targetingMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRT molecule 372NNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYATMTAEDVDLTLNWGRLSSVLPDYFIGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFKSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID exemplaryVYALPLKMLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 660 cell-targetingMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRT molecule 373NNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYATMTAEDVDLTLNWGRLSSVLPDYFIGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFKSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID exemplaryNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 661 cell-targetingMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRT molecule 374NNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYATMTAEDVDLTLNWGRLSSVLPDYFIGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTVYYCAKSMITTGFVMDSWG QGSLVTVSS SEQ ID exemplaryGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLM NO: 662 cell-targetingIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNN molecule 375VFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALIENCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQ GSLVTVS SEQ ID exemplaryDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKL NO: 663 cell-targetingLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTT molecule 376PPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSSEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTILDDLSGRSYNAITAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCH HHASAVAA SEQ IDexemplary QVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTGQG NO: 664cell-targeting LEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEDSmolecule 377 AVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSMTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARANILRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCH HHASAVAA SEQ IDexemplary KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDN NO: 665cell-targeting LFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADmolecule 378 FSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYWAKSMITTGFVMDSWGQGSLVTVSSVTEHDTLLY SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDN NO: 666 cell-targetingLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAD molecule 379FSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSNLVPMVATV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDN NO: 667 cell-targetingLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAD molecule 380FSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSQYDPVAALF SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDN NO: 668 cell-targetingLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAD molecule 381FSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSCLGGLLTMV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDN NO: 669 cell-targetingLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAD molecule 382FSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSILRGSVAHK SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDN NO: 670 cell-targetingLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAD molecule 383FSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSN VPMVATV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDN NO: 671 cell-targetingLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAD molecule 384FSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSNLVPMVATV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDN NO: 672 cell-targetingLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAD molecule 385FSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALIINCHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSIALSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATF GQGTKVEIKGILGFVFTLSEQ ID exemplary KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNNO: 673 cell-targeting LFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADmolecule 386 FSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTLTISSLQPEDEATYYCQQYNSYPYTFGQGTKLEIKGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDVHWVRQAPGQRLEWMGWLHADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARERIQLWFDYWGQGTLVTVSSNLVPMVATV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDN NO: 674 cell-targetingLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAD molecule 387FSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTLTISSLQPEDEATYYCQQYNSYPYTFGQGTKLEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSNLVPMVATV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDN NO: 675 cell-targetingLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAD molecule 388FSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVIIWVKQTPGQGLEWIGAIYPGNGDTSTNQKEKGKNFLTADKSSSTVYMQLSSLTSEDSAVYYCARSNYYGSSYVWFFDVWGAGITVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKNITMTCRASSSVSYMDWYQQKPGSSPKPWLYATSNLASGVPARESGSGSGTSYSLTISRVEAEDAATYYCQQWLSNPPTFGAGTKLE LKNLVPMVATV SEQ IDexemplary KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDN NO: 676cell-targeting LFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADmolecule 389 FSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARAQLRPNYWYEDMGAGITVTVSSGGGGSDIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKNLVPMVA TV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDN NO: 677 cell-targetingLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAD molecule 390FSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVIIWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAGSTSGSGKPGSGEGSTKGQIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLE IKNLVPMVATV SEQ IDexemplary KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDN NO: 678cell-targeting LFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADmolecule 391 FSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTDYWGQGTQVTVSSAHHSEDNLVPMVATV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDN NO: 679 cell-targetingLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAD molecule 392FSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIELTQSPSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYILSITSLQTEDVATYYCQQYWSTPTFGGGTKLEIKGSTSGSGKPGSGEGSKVQLQESGPSLVQPSQRLSITCTVSGFSLISYGVHWVRQSPGKGLEWLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTAIYFCAKTLITTGYAMDYWGQGTTVTVSSNLVPMV ATV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDN NO: 680 cell-targetingLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAD molecule 393FSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAAHHSEDPSSKAPKAPEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVNLISSIGDTYYADSVKGRFTISRDNAKNTVYLQIVINSLKPEDTAVYYCKRFRTAAQGT DYWGQGTQVTVSSNLVPMNATVSEQ ID exemplary KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNNO: 681 cell-targeting LFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADmolecule 394 FSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPASVSDVPRDLEVVAATPTSLLISWCRQRCADSYRITYGETGGNSPVQEFTVPGSWKTATISGLKPGVDYTITVYVVTHYYGWDRYSHPISINYRTGSNLVPMVATV SEQ ID exemplaryASVSDVPRDLEVVAATPTSLLISWCRQRCADSYRITYGETGGNSPV NO: 682 cell-targetingQEFTVPGSWKTATISGLKPGVDYTITVYVVTHYYGWDRYSHPISIN molecule 395YRTGSEFPKPSTPPGSSGGAPKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAANLVPMVA TV SEQ ID exemplaryKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDN NO: 683 cell-targetingLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAD molecule 396FSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIVLTQSPASLAVSLGQRATISCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPARFSGSGSGTDFSLTIHPVEEDDIAMYFCQQSRRVPYTFGGGTKLEIKGGGGSEVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYVNPFNDGTKYNEMFKGKATLTSDKSSSTAYMELSSLESEDSAVYYCARQAWGYPWGQGTLVTVSANLVPMVATV SEQ ID exemplaryNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 684 cell-targetingMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRT molecule 397NNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISTGSINAILGSVALILNCHHHASAVAAGGGGSGGDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFFFFISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID exemplaryNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 685 cell-targetingMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRT molecule 398NNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKRSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWG QGSLVTVSS SEQ ID exemplaryNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 686 cell-targetingMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRT molecule 399NNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKRSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTFPPTFGQGTKVEIKGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS SEQ ID exemplaryGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLM NO: 687 cell-targetingIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNN molecule 400VFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQY LYHPATFGQGTKVEIK SEQ IDexemplary NLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 688cell-targeting MIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTmolecule 401 NNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAGGGGSGGDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFFLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDVHWVRQAPGQRLEWMGWLHADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARERIQLWFDYWGQGTLVTVSS SEQ ID exemplaryNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 689 cell-targetingMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRT molecule 402NNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKRSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVETKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID exemplaryNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 690 cell-targetingMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRT molecule 403NNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQGLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEDSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPT FGAGTKLELK SEQ IDexemplary NLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 691cell-targeting MIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTmolecule 404 NNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAGGGGSGGQVQLVQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARAQLRPNYWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWIS NPPTFGAGTKLELK SEQ IDexemplary APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYM NO: 692cell-targeting PKKATELKHLQLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLmolecule 405 ELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTEFPKPSTPPGSSGGAPNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID exemplaryGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLM NO: 693 cell-targetingIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNN molecule 406VFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQLQEERLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT SEQ ID exemplaryMNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTS NO: 694 cell-targetingLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVN molecule 407RTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSW GQGSLVTVSS SEQ IDexemplary MNLVPMVATVKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTS NO: 695cell-targeting LLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNmolecule 408 RTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSW GQGSLVTVSS SEQ IDexemplary MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGISLLMIDSGSGD NO: 696cell-targeting NLFAVDVRGIDPEEGRFNNLRLIVERNNLNVTGFVNRTNNVFYRFAmolecule 409 DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVS SNLVPMVATV SEQ IDexemplary MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGISLLMIDSGSGD NO: 697cell-targeting NLFAVDVRGIDPEEGRFNNLRLIVERNNLNVTGFVNRTNNVFYRFAmolecule 410 DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVS SNLVPMVATV SEQ IDexemplary MGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLL NO: 698cell-targeting MIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTmolecule 411 NNVFYRFADFSHVTFPGTTAVTLSGDSSYTFLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEAERFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNIKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWG QGSLVTVSS SEQ ID exemplaryMDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAP NO: 699 cell-targetingKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHY molecule 412TTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSSEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNC HHHASAVAA SEQ IDexemplary MQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQ NO: 700cell-targeting GLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEmolecule 413 DSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLREVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN CHHHASAVAA SEQ IDexemplary MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGD NO: 701cell-targeting NLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 414 DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSNLVPMVAT V SEQ ID exemplaryMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGD NO: 702 cell-targetingNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 415DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDETLTISSLQPEDFATYYCQQYLYHPATF GQGTKVEIKGILGFVFTLSEQ ID exemplary MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNO: 703 cell targeting NLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 416 DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDETLTISSLQPEDFATYYCQQYLYHPATF GQGTKVEIKGILGFVFTLSEQ ID exemplary MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNO: 704 cell-targeting NLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 417 DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGFKLEIKGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDVHWVRQAPGQRLEWMGWLHADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARERIQLWFDYWGQGTLVTVSSNLVPMVATV SEQ ID exemplaryMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGD NO: 705 cell targetingNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 418DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFFFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSNLVPMVATV SEQ ID exemplaryQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQG NO: 706 cell-targetingLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEDS molecule 419AVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCH HHASAVAA SEQ IDexemplary MQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQ NO: 707cell-targeting GLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEmolecule 420 DSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN CHHHASAVAA SEQ IDexemplary MDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAP NO: 708cell-targeting KLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYmolecule 421 TTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSSEFPKPSTPPGSSGGAPGILGFVFFLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNC HHHASAVAAKDEL SEQ IDexemplary MDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAP NO: 709cell-targeting KLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYmolecule 422 TTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSSEFPKPSTPPGSSGGAPGILGFVFFLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNC HHHASAVAAKDEL SEQ IDexemplary MDIELTQSPSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGNAPRL NO: 710cell-targeting LISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSmolecule 423 TPTFGGGTKLEIKGSTSGSGKPGSGEGSKVQLQESGPSLVQPSQRLSITCTVSGFSLISYGVHWVRQSPGKGLEWLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFEKMNSLQADDTAIYFCAKTLITTGYAMDYWGQGTTVTVSSEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAV AAKDEL SEQ ID exemplaryMDIELTQSPSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGNAPRL NO: 711 cell-targetingLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWS molecule 424TPTFGGGTKLEIKGSTSGSGKPGSGEGSKVQLQESGPSLVQPSQRLSITCTVSGFSLISYGVHWVRQSPGKGLEWLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFEKMNSLQADDTAIYFCAKTLITTGYAMDYWGQGTTVTVSSEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAV AAKDEL SEQ ID exemplaryMDIVMTQAAPSIPVTPGESVSISCRSSKSLNSNGNTYLYWFLQRPG NO: 712 cell-targetingQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYC molecule 425MQHLEYPFTFGAGTKLELKGSTSGSGKPGSGEGSEVQLQQSGPELIKPGASVKMSCKASGYTFTSYVMHWVKQKPCQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGTYYYGSRVFDYWGQGTTLTVSSAEFPKPSTPPGSSGGAPGILGFVFTLEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSV ALILNCHHHASAVAAKDELSEQ ID exemplary MDIVMTQAAPSIPVTPGESVSISCRSSKSLNSNGNTYLYWFLQRPG NO: 713cell-targeting QSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCmolecule 426 MQHLEYPFTFGAGTKLELKGSTSGSGKPGSGEGSEVQLQQSGPELIKPGASVKMSCKASGYTFTSYVMHWVKQKPCQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGTYYYGSRVFDYWGQGTTLTVSSAEFPKPSTPPGSSGGAPGILGFVFTLEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSV ALILNCHHHASAVAAKDELSEQ ID exemplary MDIQLTQSPLSLPVTLGQPASISCRSSQSLVHRNGNTYLHWFQQRPG NO: 714cell-targeting QSPRLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSmolecule 427 QSSHVPPTFGAGTRLEIKGSTSGSGKPGSGEGSTKGQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGVNWIKQAPGQGLQWMGWINPNTGEPTFDDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCSRSRGKNEAWFAYWGQGTLVTVSSEEPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTVVDSLNVIRSAIGTPLQTISSGGTSLMIDSGSGDNLFAVDVRGIDPEEGRFNNIALIVERNNLYVTGFVNRFVNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLFFSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVA LILNCHHHASAVAAKDEL SEQ IDexemplary MDIQLTQSPLSLPVTLGQPASISCRSSQSLVHRNGNTYLHWFQQRPG NO: 715cell-targeting QSPRLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSmolecule 428 QSSHVPPTFGAGTRLEIKGSTSGSGKPGSGEGSTKGQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGVNWIKQAPGQGLQWMGWINPNTGEPTFDDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCSRSRGKNEAWFAYWGQGTLVTVSSEEPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTVVDSLNVIRSAIGTPLQTISSGGTSLMIDSGSGDNLFAVDVRGIDPEEGRFNNIALIVERNNLYVTGFVNRFVNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLFFSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVA LILNCHHHASAVAAKDEL SEQ IDexemplary MEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQ NO: 716cell-targeting RELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAmolecule 429 VYYCKRFRTAAQGTDYWGQGTQVTVSSAHHSEDPSSKAPKAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLFQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHASAVAAKDEL SEQ ID exemplaryMEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQ NO: 717 cell-targetingRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA molecule 430VYYCKRFRTAAQGTDYWGQGTQVTVSSAHHSEDPSSKAPKAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLFQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHASAVAAKDEL SEQ ID exemplaryMEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQ NO: 718 cell-targetingRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA molecule 431VYYCKRFRTAAQGTDYWGQGTQVTVSSAHHSEDPSSKAPKAPGILGFVFTLGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHASAVAAKDEL SEQ ID exemplaryMEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQ NO: 719 cell-targetingRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTA molecule 432VYYCKRFRTAAQGTDYWGQGTQVTVSSAHHSEDPSSKAPKAPGILGFVFTLGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHASAVAAKDEL SEQ ID exemplaryMAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFY NO: 720 cell-targetingMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVI molecule 433VLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAKDEL SEQ ID exemplaryMAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFY NO: 721 cell-targetingMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVI molecule 434VLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAKDEL SEQ ID exemplaryMQVQLVQSGAELVKPGASVKMSCKASGYITTSYNMHWVKQTPGQ NO: 722 cell-targetingGLEWIGATYPGNGDTSYNQKFKGKATETADKSSSTAYNIQLSSLTSE molecule 435DSAVYYCARAQLRPNYWYFDVWGAGTTVTVSSGSTSGSGKPGSGEGSDIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLASGYPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRIGMQINRHSETTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCH HHASAVAA SEQ IDexemplary MQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQ NO: 723cell-targeting GLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEmolecule 436 DSAVYYCARAQLRPNYWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN CHHHASAVAA SEQ IDexemplary MQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQ NO: 724cell-targeting GLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEmolecule 437 DSAVYYCARAQLRPNYWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN CHHHASAVAA SEQ IDexemplary MQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQ NO: 725cell-targeting GLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEmolecule 438 DSAVYYCARAQLRPNYWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN CHHHASAVAA SEQ IDexemplary MQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGR NO: 726cell-targeting GLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEmolecule 439 DSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAGSTSGSGKPGSGEGSTKGQIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN CHHHASAVAA SEQ IDexemplary MEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGK NO: 727cell-targeting GLEWVSTISWNSGSIGYADSVKGRFTISDNAKKSLYLQMSLRAE molecule 440DTALYYCAKDIQYGNYYYGMDVWGTGTTVTVSSGSTSGSGKPGSGEGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNC HHHASAVAA SEQ IDexemplary MEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRL NO: 728cell-targeting LIYDASNRATGIPARFSGSGSGTDFFLTISSLEPEDFAVYYCQQRSNWmolecule 441 PITFGQGTRLEIKGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRERQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINA ILGSVALILNCHHHASAVAASEQ ID exemplary MQIVLSQSPAILSASPGEKVTMTCRASSVSYMHWYQQKPGSSPKP NO: 729cell-targeting WIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFmolecule 442 NPPTFGAGTKLELKSGGGGSGGGGSGGGGSGGGGSGGGGSQAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTLTTVTVSEFPKPSTPPGSSGGILGFVFTLGAPKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTNWGRLSSVLPDYHGQDSVRVGRISFG SINAILGSVALILNCHHHASAVAASEQ ID exemplary MQAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQ NO: 730cell-targeting GLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEmolecule 443 DSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSGSTSCLSGKPGSGEGSQIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKSEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSHLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCH HHASAVAA SEQ IDexemplary ASVSDVPRDLEVVAATPTSLLISWCRQRCADSYRITYGETGGNSPV NO: 731cell-targeting QEFTVPGSWKTATISGLKPGVDYTITVYVVTHYYGWDRYSHPISINmolecule 444 YRTGSEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLSVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTADALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAA SEQ ID exemplaryMQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQ NO: 732 cell-targetingGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSE molecule 445DSAVYYCARSNYYGSSYVWFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAGTGDALVALPEGESVRIADIVPGARPNSDNAIDLKVLDRHGNPVLADRLFHSGEHPVYTVRTVEGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAVIQRSAFSVDCAGFARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYYAKVASVTDAGVQPVYSLRVDTADHAFITNGFVSHATGLTLINSGLTTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVPALWQLQ SEQ ID exemplaryMQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQ NO: 733 cell-targetingGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSE molecule 446DSAVYYCARSNYYGSSYVWFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAGTGDALVALPEGESVRIADIVPGARPNSDNAIDLKVLDRHGNPVLADRLFHSGEHPVYTVRTVEGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAVIQRSAFSVDCAGFARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYYAKVASVTDAGVQPVYSLRVDTADHAFITNGFVSHATGLTLINSGLTTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVPALWQLQ SEQ ID exemplaryMQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQ NO: 734 cell-targetingGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSE molecule 447DSAVYYCARSNYYGSSYVWFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTLKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILN CHHHASAVAAKDEL SEQ IDexemplary MKEFTLDFSTAKTYVDSINVIRSAIGTPLQTISSGGTSLLMIDSGSGD NO: 735cell-targeting NLFAVDVRGIDPEEGRGNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 448 DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLMQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVHWVKQTPGQGLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEDSAVYYCARSNYYGSSYVWFFDNIVGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISN PPTFGAGTKLELK SEQ IDexemplary MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGD NO: 736cell-targeting NLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 449 DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIELTQSPSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPTFGGGTKLEIKGSTSGSGKPGSGEGSKVQLQESGPSLVQPSQRLSITCTVSGFSLISYGVHWVRQSPGKGLEWLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTAIYFCAKTLITTGYAMDYWGQGTTVT VSS SEQ ID exemplaryMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGD NO: 737 cell-targetingNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 450DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIELTQSPSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPTFGGGTKLEIKGSTSGSGKPGSGEGSKVQLQESGPSLVQPSQRLSITCTVSGFSLISYGVHWVRQSPGKGLEWLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTAIYFCAKTLITTGYAMDYWGQGTTVT VSS SEQ ID exemplaryMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGD NO: 738 cell-targetingNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 451DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIQMTQSPSSLSASVGDRVITITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMD VWGQGTLVTVSS SEQ IDexemplary MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGD NO: 739cell-targeting NLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 452 DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIQMTQSPSSLSASVGDRVITITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMD VWGQGTLVTVSS SEQ IDexemplary MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGD NO: 740cell-targeting NLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 453 DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPGILGTVFTLDIVMTQAAPSIPVTPGESVSISCRSSKSLLNSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGAGTKLELKGSTSGSGKPGSGEGSEVQLQQSGPELIKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGTYYYGSR VFDYWGQGTTLTVSS SEQ IDexemplary MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGD NO: 741cell-targeting NLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 454 DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPGILGTVFTLDIVMTQAAPSIPVTPGESVSISCRSSKSLLNSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGAGTKLELKGSTSGSGKPGSGEGSEVQLQQSGPELIKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGTYYYGSR VFDYWGQGTTLTVSS SEQ IDexemplary MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGD NO: 742cell-targeting NLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 455 DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIQLTQSPLSLPVTLGQPASISCRSSQSLVHRNGNTYLHWFQQRPGQSPRLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSSHVPPTFGAGTRLEIKGSTSGSGKPGSGEGSTKGQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGVNWIKQAPGQGLQWMGWINPNTGEPTFDDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCSRSRGKNEAW FAYWGQGTLVTVSS SEQ IDexemplary MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGD NO: 743cell-targeting NLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 456 DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIQLTQSPLSLPVTLGQPASISCRSSQSLVHRNGNTYLHWFQQRPGQSPRLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSSHVPPTFGAGTRLEIKGSTSGSGKPGSGEGSTKGQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGVNWIKQAPGQGLQWMGWINPNTGEPTFDDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCSRSRGKNEAW FAYWGQGTLVTVSS SEQ IDexemplary MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGD NO: 744cell-targeting NLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 457 DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAAHHSEDPSSKAPKAPGILGFVFTLEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCK RFRTAAQGTDYWGQGTQVTVSSSEQ ID exemplary MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNO: 745 cell-targeting NLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 458 DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC KRFRTAAQGTDYWGQGTQVTVSSSEQ ID exemplary MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNO: 746 cell-targeting NLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 459 DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAAHHSEDPSSKAPKAPGILGFVFTLEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCK RFRTAAQGTDYWGQGTQVTVSSSEQ ID exemplary MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNO: 747 cell-targeting NLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAmolecule 460 DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIVLTQSPASLAVSLGQRATISCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPARFSGSGSGTDFSLTIHPVEEDDIAMYFCQQSRRVPYTFGGGTKLEIKGGGGSEVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYVNPFNDGTKYNEMFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARQAWGYPWGQGTLVTVSA SEQ ID exemplaryMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGD NO: 748 cell-targetingNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 461DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIVLTQSPASLAVSLGQRATISCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPARFSGSGSGTDFSLTIHPVEEDDIAMYFCQQSRRVPYTFGGGTKLEIKGGGGSEVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYVNPFNDGTKYNEMFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARQAWGYPWGQGTLVTVSA SEQ ID exemplaryMKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGD NO: 749 cell-targetingNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFA molecule 461DFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPGILGFVFTLDIVLTQSPASLAVSLGQRATISCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPARFSGSGSGTDFSLTIHPVEEDDIAMYFCQQSRRVPYTFGGGTKLEIKGGGGSEVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYVNPFNDGTKYNEMFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARQAWGYPWGQGTLVTVSA NLVPMVATV

1. A cell-targeting molecule comprising i) a Shiga toxin effectorpolypeptide having a carboxy-terminus and comprising: a) an embedded orinserted, heterologous, CD8+ T-cell epitope; b) a disruption of at leastone, endogenous, B-cell and/or CD4+ T-cell epitope region which does notoverlap with the embedded or inserted, heterologous, CD8+ T-cellepitope; c) Shiga toxin A1 fragment region having a carboxy-terminus;and d) a disrupted furin-cleavage motif at the carboxy-terminus of theA1 fragment region; ii) a binding region capable of specifically bindingat least one extracellular target biomolecule, and iii) a heterologous,CD8+ T-cell epitope cargo which is not embedded or inserted in the Shigatoxin A1 fragment region, wherein the CD8+ T-cell epitope cargo ispositioned carboxy-terminal to the carboxy terminus of the Shiga toxinA1 fragment region.
 2. The cell-targeting molecule of claim 1, whereinthe Shiga toxin effector polypeptide is capable of delivering the CD8+T-cell epitope cargo from an early endosomal compartment of a cell inwhich the Shiga toxin effector polypeptide is present to a MHC class Imolecule of the cell, and wherein administration of the cell-targetingmolecule to a cell physically coupled with an extracellular targetbiomolecule bound by the binding region, the cell-targeting moleculeinternalizes into the cell resulting in the presentation of the CD8+T-cell epitope cargo complexed with a MHC class I molecule on a cellularsurface of the cell.
 3. The cell-targeting molecule of claim 2, whereinthe CD8+ T-cell epitope cargo is fused to the Shiga toxin effectorpolypeptide or the binding region.
 4. The cell-targeting molecule ofclaim 3, wherein the cell-targeting molecule comprises or consists of asingle-chain polypeptide comprising (i) the Shiga toxin effectorpolypeptide, (ii) the binding region, and (iii) the CD8+ T-cell epitopecargo.
 5. (canceled)
 6. The cell-targeting molecule of claim 1, whichcomprises a molecular moiety associated with the carboxy-terminus of theShiga toxin effector polypeptide.
 7. The cell-targeting molecule ofclaim 6, wherein the molecular moiety comprises the binding region. 8.The cell-targeting molecule of claim 6, wherein the molecular moiety iscytotoxic.
 9. The cell-targeting molecule of claim 7, wherein themolecular moiety comprises an amino acid residue to which the Shigatoxin effector polypeptide is linked.
 10. The cell-targeting molecule ofclaim 9, wherein the molecular moiety and the Shiga toxin effectorpolypeptide are fused forming a continuous polypeptide.
 11. Thecell-targeting molecule of claim 1, wherein the binding region comprisesa: single-domain antibody fragment, single-chain variable fragment,antibody variable fragment, complementary determining region 3 fragment,constrained FR3-CDR3-FR4 polypeptide, Fd fragment, antigen-bindingfragment, Armadillo repeat polypeptide, fibronectin-derived 10thfibronectin type Ill domain, tenascin type III domain, ankyrin repeatmotif domain, low-density-lipoprotein-receptor-derived A-domain,lipocalin, Kunitz domain, Protein-A-derived Z domain, gamma-Bcrystallin-derived domain, ubiquitin-derived domain, Sac7d-derivedpolypeptide, Fyn-derived SH2 domain, miniprotein, C-type lectin-likedomain scaffold, or a genetically manipulated counterparts of any of theforegoing which retains extracellular target biomolecule binding. 12.The cell-targeting molecule of claim 11, wherein the Shiga toxineffector polypeptide comprises or consists of a sequence that is atleast 75%, 85%, 95%, 96%, 97%, 98%/o or more identical to: (i) aminoacids 75 to 251 of any one of SEQ ID NOs: 1-6; (ii) amino acids 1 to 241of any one of SEQ ID NOs: 1-18; (iii) amino acids 1 to 251 of any one ofSEQ ID NOs: 1-6; or (iv) amino acids 1 to 261 of any one of SEQ ID NOs:1-3.
 13. (canceled)
 14. The cell-targeting molecule of claim 12, whereinthe disrupted furin-cleavage motif comprises a mutation, relative to awild-type Shiga toxin A Subunit, the mutation altering at least oneamino acid residue in a region natively positioned at 248-251 of any oneof SEQ ID NOs: 1-2 and, or at 247-250 of any one of SEQ ID NOs: 3 and7-18.
 15. (canceled)
 16. The cell-targeting molecule of claim 15,wherein the amino acid residue substitution is of an arginine residuewith a non-positively charged, amino acid residue selected from:alanine, glycine, proline, serine, threonine, aspartate, asparagine,glutamate, glutamine, cysteine, isoleucine, leucine, methionine, valine,phenylalanine, tryptophan, and tyrosine.
 17. The cell-targeting moleculeof claim 11, wherein the extracellular target biomolecule: CD20, CD22,CD40, CD74, CD79, CD25, CD30, HER2/neu/ErbB2, EGFR, EpCAM, EphB2,prostate-specific membrane antigen, Cripto, CDCP1, endoglin, fibroblastactivated protein, Lewis-Y, CD19, CD21, CSI/SLAMF7, CD33, CD52, CD133,CEA, gpA33, mucin, TAG-72, tyrosine-protein kinase transmembranereceptor, carbonic anhydrase IX, folate binding protein, gangliosideGD2, ganglioside GD3, ganglioside GM2, ganglioside Lewis-Y2, VEGFR,Alpha V beta3, Alpha5beta1, ErbB1/EGFR, Erb3, c-MET, IGF1R, EphA3,TRAIL-R1, TRAIL-R2, RANK, FAP, tenascin, CD64, mesothelin, BRCA1,MART-1/MelanA, gp100, tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3,GAGE-1/2, BAGE, RAGE, NY-ESO-I, CDK-4, beta-catenin, MUM-I, caspase-8,KIAA0205, HPVE6, SART-1, PRAME, carcinoembryonic antigen, prostatespecific antigen, prostate stem cell antigen, human aspartyl(asparaginyl) beta-hydroxylase, EphA2, HER3/ErbB-3, MUC1, MART-1/MelanA,gp100, tyrosinase associated antigen, HPV-E7, Epstein-Barr virusantigen, Bcr-Abl, alpha-fetoprotein antigen, 17-A1, bladder tumorantigen, SAIL, CD38, CD15, CD23, CD45, CD53, CD88, CD129, CD183, CD191,CD193, CD244, CD294, CD305, C3AR, FceRIa, IL-1R, galectin-9, mrp-14,NKG2D, PD-L1, Siglec-8, Siglec-10, CD49d, CD13, CD44, CD54, CD63, CD69,CD123, TLR4, FceRIa, IgE, CD107a, CD203c, CD14, CD68, CD80, CD86, CD105,CD115, F4/80, ILT-3, galectin-3, CD11a-c, GITRL, MHC class I molecule,MHC class II molecule, CD284, CD107-Mac3, CD195, HLA-DR, CD16/32, CD282,CD11c, or any immunogenic fragment of any of the foregoing.
 18. Thecell-targeting molecule of claim 2, wherein the Shiga toxin effectorpolypeptide is capable of exhibiting one or more Shiga toxin effectorfunctions in addition to delivery of the CD8+ T-cell epitope cargo froman early endosomal compartment of a cell in which the Shiga toxineffector polypeptide is present to a MHC class I molecule of the cell.19-20. (canceled)
 21. The cell-targeting molecule of claim 2, wherebyadministration of the cell-targeting molecule to a cell physicallycoupled with an extracellular target biomolecule of the binding regionresults in death of the cell. 22-30. (canceled)
 31. The cell-targetingmolecule of claim 1, in the form of a pharmaceutically acceptable saltor solvate.
 32. A pharmaceutical composition comprising thecell-targeting molecule of claim 1 and at least one pharmaceuticallyacceptable excipient or carrier.
 33. A polynucleotide capable ofencoding the cell-targeting molecule of claim 4, or a complementthereof. 34-36. (canceled)
 37. A method of delivering a CD8+ T-cellepitope to an intracellular MHC class I molecule of a cell, the methodcomprising the step of contacting the cell with the cell-targetingmolecule claim 1 and/or the pharmaceutical composition of claim 32.38-39. (canceled)
 40. A method of treating a disease, disorder, orcondition in a patient, the method comprising the step of administeringto a patient in need thereof a therapeutically effective amount of thecell-targeting molecule of claim 1 or the pharmaceutical composition ofclaim
 32. 41-53. (canceled)